Gram-negative alkaliphilic microorganisms

ABSTRACT

Gram-negative bacteria, which are obligate alkaliphiles, have been isolated from samples of soil, water and sediment and a number of other sources obtained from in and around soda lakes. These bacteria have been analyzed according to the principles of numerical taxonomy with respect to each other, as well as to a variety of known bacteria. In addition, these bacteria are further circumscribed by an analysis of various chemotaxonomic characteristics. The bacteria produce various alkali-tolerant enzymes which may be used in various industrial processes requiring such enzymatic activity in a high pH environment.

This application is a divisional of U.S. Ser. No. 08/122,745, filed Sep.16, 1993, now U.S. Pat. No. 5,459,062, which is a continuation-in-partof U.S. Ser. No. 07/046,878, filed Apr. 8, 1993, which is acontinuation-in-part of U.S. Ser. No. 07/903,786, filed Jun. 24, 1992,now U.S. Pat. No. 5,401,657, which is a continuation-in-part of U.S.Ser. No. 07/719,307, filed Jun. 24, 1991, now abandoned, which is acontinuation-in-part of U.S. Ser. No. 07/562,863, filed Aug. 6, 1990,now abandoned, all of which are incorporated herein by reference.

The present invention is in the field of microbiology and moreparticularly in the field of alkaliphilic microorganisms.

BACKGROUND OF THE INVENTION

Alkaliphiles are defined as organisms which exhibit optimum growth in analkaline pH environment, particularly in excess of pH 8, and generallyin the range between pH 9 and 10. Alkaliphiles may also be found livingin environments having a pH as high as 12. Obligate alkaliphiles areincapable of growth at neutral pH.

Alkaliphiles may be found in such everyday environments as garden soil,presumably due to transient alkaline conditions caused by biologicalactivity such as ammonification, sulphate reduction or photosynthesis. Amuch richer source of a greater variety of alkaliphilic organisms may befound in naturally occurring, stable alkaline environments such as sodalakes.

A more detailed study of soda lakes and alkaliphilic organisms ingeneral is provided in Grant, W. D., Mwatha, W. E. and Jones, B. E.((1990) FEMS Microbiology Reviews, 75, 255-270), the text of which ishereby incorporated by reference. Lists of alkaline soda lakes may befound in the publications of Grant, W. D. and Tindall, B. J. in Microbesin Extreme Environments, (eds. R. A. Herbert and G. A. Codd); AcademicPress, London, (1986) , pp. 22-54; and Tindall, B. J. in HalophilicBacteria, Volume 1, (ed. F. Rodriguez-Valera); CRC Press Inc., BocaRaton, Fla., (1988), pp. 31-70, both texts are also hereby incorporatedby reference.

Alkaliphiles, the majority of which are Bacillus species, have beenisolated from non-saline environments and are discussed by Horikoshi, K.and Akiba, T. in Alkalophilic Microorganisms (Springer-Verlag, Berlin,Heidelberg, N.Y., (1982)) . However, alkaliphilic organisms from salineand alkaline environments such as lakes are not discussed therein.Strictly anaerobic bacteria from alkaline, hypersaline, environmentshave been recently described by Shiba, H. in Superbugs (eds. K.Horikoshi and W. D. Grant); Japan Scientific Societies Press, Tokyo andSpringer-Verlag, Berlin, Heidelberg, N.Y., (1991), pp. 191-211; and byNakatsugawa. N., ibid, pp. 212-220.

Soda lakes, which may be found in various locations around the world,are caused by a combination of geological, geographical and climaticconditions. They are characterized by the presence of large amounts ofsodium carbonate (or complexes thereof) formed by evaporativeconcentration, as well as by the corresponding lack of Ca²⁺ and Mg²⁺which would remove carbonate ions as insoluble salts. Other salts suchas NaCl may also concentrate resulting in environments which are bothalkaline and saline.

Despite this apparently harsh environment, soda lakes are neverthelesshome to a large population of prokaryotes, a few types of which maydominate as permanent or seasonal blooms. The organisms range fromalkaliphilic cyanobacteria to haloalkaliphilic archaeobacteria.Moreover, it is not unusual to find common types of alkaliphilicorganisms inhabiting soda lakes in various widely dispersed locationsthroughout the world such as in the East African Rift Valley, in thewestern U.S., Tibet, China and Hungary. For example, natronobacteriahave been isolated and identified in soda lakes located in China (Wang,D. and Tang, Q., "Natronobacterium from Soda Lakes of China" in RecentAdvances in Microbial Ecology (Proceedings of the 5th InternationalSymposium on Microbial Ecology, eds. T. Hattori et al.); JapanScientific Societies Press, Tokyo, (1989), pp. 68-72) and in the westernU.S. (Morth, S. and Tindall, B. J. (1985) System. Appl. Microbiol., 6,247-250). Natronobacteria have also been found in soda lakes located inTibet (W. D. Grant, unpublished observations) and India (Upasani, V. andDesai, S. (1990) Arch. Microbiol., 154, pp. 589-593).

Alkaliphiles have already made an impact in the application ofbiotechnology for the manufacture of consumer products. Alkali-tolerantenzymes produced by alkaliphilic microorganisms have already found usein industrial processes and have considerable economic potential. Forexample, these enzymes are currently used in detergent compositions andin leather tanning, and are foreseen to find applications in the food,waste treatment and textile industries. Additionally, alkaliphiles andtheir enzymes are potentially useful for biotransformations, especiallyin the synthesis of pure enantiomers.

SUMMARY OF THE INVENTION

The present invention provides pure cultures of novel aerobic,Gram-negative alkaliphilic bacteria. These bacteria have been isolatedfrom samples of soil, water, sediment and a number of other sources, allof which were obtained from in and around alkaline soda lakes. Thesealkaliphiles have been analyzed according to the principles of numericaltaxonomy with respect to each other and also to a variety of knownbacteria in order to confirm their novelty. In addition, these bacterialtaxa are further circumscribed by an analysis of various chemotaxonomiccharacteristics.

The present invention also provides data as to the composition of theenvironments from which the samples containing the microorganisms wereobtained, as well as the media required for their efficient isolationand culturing such that one of ordinary skill may easily locate such anenvironment and be able to isolate the organisms of the presentinvention by following the procedures described herein.

It is also an object of the present invention to provide microorganismswhich produce useful alkali-tolerant enzymes, as well as methods forobtaining substantially pure preparations of these enzymes. Theseenzymes are capable of performing their functions at high pH which makesthem uniquely suited for applications requiring such extreme conditions.For example, alkali-tolerant enzymes may be employed in detergentcompositions, in leather tanning and in the food, waste treatment andtextile industries, as well as for biotransformations such as theproduction of pure enantiomers.

The genes encoding these alkali-tolerant enzymes may be isolated, clonedand brought to expression in compatible expression hosts to provide asource of larger volumes of enzyme products which may be, if desired,more easily purified and used in various industrial applications, shouldthe wild-type strain fail to produce sufficient amounts of the desiredenzyme, or does not ferment well.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Simplified dendrogram showing clusters (phenons) obtained withthe S_(G) coefficient and Unweighted Average Linkage procedure.

FIG. 2. Simplified dendrogram showing clusters (phenons) obtained withthe S_(J) coefficient and Unweighted Average Linkage procedure.

FIG. 3. Simplified dendrogram obtained with the S_(G) coefficient andUnweighted Average Linkage procedure using the derived minimumdiscriminatory tests.

FIG. 4. Unrooted phylogenetic tree showing the relationship of 3alkaliphilic bacteria to representatives of the gamma-Proteobacteria.

DETAILED DESCRIPTION OF THE INVENTION Sampling

Several hundreds of strains of bacteria have been isolated from samplesof soil, water, sediment and a number of other sources in and aroundalkaline lakes. These samples were obtained as part of an investigationover a period of three years. The isolated bacteria are non-phototrophiceubacteria. Up until now, such bacteria have not been wellcharacterized.

The samples were collected in sterile plastic bags. Sampling wasconducted at lakes Elmenteita, Nakuru, Bogoria, Crater (Sonachi), LittleNaivasha (Oloidien), Magadi, and Little Magadi (Nasikie Engida), all ofwhich are located in Kenya, East Africa. Alkaline soda lakes havingsimilar environments may also be found in Tibet, China, Hungary and thewestern U.S. At each sampling site, physical parameters such as pH,conductivity and temperature were measured as well as the physicalappearance of the site and the sample. Some of the samples were treatedlocally within 36 hours of collection of the sample but the majoritywere examined offsite, several weeks after collection.

Table 1 lists various strains which have been isolated. The strains arelisted according to the location from which the sample was taken, thephysical appearance of the sample itself and a reference to Table 2which provides the chemical analysis of the lake water samples.

Table 3 provides a list of the isolated strains arranged according tothe results of the numerical taxonomic analysis. Furthermore, Table 3provides physical properties of the sample, in particular thetemperature, conductivity and alkaline pH, as well as the numerousisolation media required for obtaining pure cultures of the newbacteria. These media are letter coded with reference to Appendix A.

Tables 1, 2 and 3 provide data from which the environment of thesampling locations may be characterized. The chemical and physicalanalysis of the samples confirm the presence of alkaline pH, as well asthe presence of unusually high levels of Na₂ CO₃, coupled with lowlevels of Ca²⁺ and Mg²⁺.

No chemical analysis is available for mud samples. Furthermore, nochemical analysis is available for a few samples (see Table 1). However,other samples taken at the same location have been analyzed and aredescribed in Tables 1-3. It is known that the basic environments of sodalakes are stable with respect to their pH and ionic composition.Moreover, the microbial populations found at these sites remain largelystable. Thus, it is to be expected that despite the lack of a chemicalanalysis of certain samples, the environment from which the bacteriawere obtained may nonetheless be determined from the data presented inTables 1-3.

The fresh soda-lake water samples were plated out on an alkalinenutrient medium (Medium A) soon after collection. Microscopic inspectionshowed an unexpectedly high diversity of bacterial types. Consideringthe extremely alkaline nature of the environment, viable counts showedunexpectedly high numbers of organotrophic bacteria, in the range of 10⁵-10⁶ colony forming units per ml. The samples were stored either cooledor at ambient temperatures. After a few weeks' storage, the totalnumbers of bacteria in the sample rose, whereas the diversity of typesdecreased.

                  TABLE 1    ______________________________________    Alkaliphilic Strains Arranged According to Their Place of Origin                                        ANAL-               SAMPLE      SAMPLE       YSIS    STRAINS    LOCATION    APPEARANCE   (Table 2)    ______________________________________    1E.1, 2E.1,               Lake Elmen- Mud from dried                                        N.R.    4E.1, 5E.1,               teita (east bay)                           up lake bed    35E.2, 36E.2,    37E.2, 38E.2    wE.5, wEl1,               Lake Elmen- Sediment and N.T.    wEl2       teita (east bay)                           water, littoral                           zone    39E.3, 40E.3,               Lake Elmen- Mud and water                                        1    41E.3, 42E.3,               teita (east bay)                           littoral zone.    44E.3, 53E.4,          Spirulina scum    56E.4    45E.3, 47E.3,               Lake Elmen- Brown water  2    57E.4      teita swamp,                           and sediment               south-      east arm    48E.3, 58E.4               Lake Elmen- Grey mud     2               teita swamp,               south-    59E.4      Lake Elmen- Water and sandy                                        3               teita, (north-                           sediment, littoral               west bay)   zone    16N.1, 17N.1,               Lake Nakuru,                           Mud and water,                                        N.T.    18N.1, 19N.1,               north beach littoral zone.    20N.1, 26N.1,               between Hippo    28N.1, wN1,               Point and Njoro    wN2, wNk1, Point.    wNk2    49N.3, 50N.3,               Lake Nakuru,                           Water column,                                        4    61N.4      north beach littoral zone.               between Hippo               Point and Njoro               Point.    51N.3, 52N.3               Lake Nakuru,                           Lake sediment,                                        4               north beach littoral zone.               between Hippo               Point and Njoro               Point.    63N.4      Lake Nakuru;                           Mud and water                                        N.T.               water hole, SW               salt flats    6B.1, 7B.1,               Lake Bogoria,                           Mud and water,                                        N.T.    8B.1, 9B.1,               northern mud                           littoral zone    10B.1, 24B.1,               flats    25B.1, wB1,    wB2, wB4, wB5    wBn4    64B.4      Lake Bogoria,                           Dried crust of                                        N.R.               northern mud                           soda mud               flats    65B.4      Lake Bogoria,                           Mud at water 5               northern mud                           line               flats    wBs4       Lake Bogoria,                           Mud and water,                                        N.T.               south-west  littoral zone               shore    11C.1, 12C.1,               Crater Lake,                           Mud and water,                                        N.T.    29C.1,     North point littoral zone    73aC.4, 73bC.4,               Crater Lake,             6    74C.4      North point    75C.4      Crater Lake,                           Soda-mud, shore                                        N.R.               North point line    77LN.4, 78LN.4               Little Lake Water column 7               Naivasha,   and sediment               south shore    21M.1, 22M.1,               Lake Magadi,                           Mud and water                                        N.T.    27M.1      causeway upper               western arm    92LM.4, 94LM.4               Little Lake Spring water and                                        8               Magadi, north-                           sediment               west springs    ______________________________________     N.T. = not tested     N.R. = not relevant

                                      TABLE 2    __________________________________________________________________________    Chemical Analysis of Kenyan Soda Lake Waters           Na.sup.+               K.sup.+                   Ca.sup.2+                       Mg.sup.2+                           SiO.sub.2                               PO.sub.4.sup.3-                                   Cl.sup.-                                       SO.sub.4.sup.2-                                           CO.sub.3.sup.2-                                               TON*    ANALYSIS           (mM)               (mM)                   (mM)                       (mM)                           (mM)                               (mM)                                   (mM)                                       (mM)                                           (mM)                                               (mM)                                                   TA§    __________________________________________________________________________    1      196 3.58                   0.07                       b.l.d.                           2.91                               0.03                                   65.1                                       2.0 68.0                                               0.8 119    2      140 3.32                   0.48                       0.13                           1.85                               0.02                                   46.8                                       1.7 32.0                                               1.2  86    3      167 3.32                   0.06                       b.l.d.                           3.10                               0.03                                   51.8                                       1.7 64.0                                               2.2 103    4      326 5.63                   0.15                       b.l.d.                           3.25                               0.15                                   57.5                                       0.5 198.3                                               1.9 259    5      735 5.50                   0.21                       0.01                           2.23                               0.09                                   100.9                                       1.0 476.7                                               0.9 612    6      140 8.95                   0.06                       0.01                           2.13                               0.04                                   12.4                                       0.8 90.0                                               1.1 133    7      8.7 1.79                   0.28                       0.65                           1.02                               0.003                                   4.8 0.5 <10.0                                               <0.07                                                    18    8      483 4.35                   0.03                       0.03                           0.64                               0.08                                   157.8                                       1.7 166.0                                               1.2 105    __________________________________________________________________________     b.l.d. = below the limits of detection     *TON = Total Organic Nitrogen     §TA = Total Alkalinity in milliequivalents/liter

                                      TABLE 3    __________________________________________________________________________    Origin of the Strains Arranged by Cluster*               SAMPLE                           Temp.                               Conductivity                                      ISOLATION    CLUSTER          STRAIN               LOCATION                      pH   °C.                               mS/cm  MEDIUM    __________________________________________________________________________    1     1E.1.sup.CT               Elmenteita                       9.5 35  n.t.   A    1     2E.1 Elmenteita                       9.5 35  n.t.   A    1     wB2  Bogoria                      n.t. n.t.                               n.t.   A    1     wB5  Bogoria                      n.t. n.t.                               n.t.   A    1     wBs4 Bogoria                      10.5 n.t.                               19     A    1     10B.1               Bogoria                      10.5 36  45     A    1     20N.1               Nakuru 10.5 36  30-40  A    1     27M.1               Magadi 11.0 36  100    A    1     Comamonas terrigena.sup.T (NCIMB 8193)                                      --    1     wNk2 Nakuru 10.5 n.t.                               19     A    1     Pseudomonas putida.sup.T (NCIMB 9494)                                      --    2     39E.3               Elmenteita                      10-10.5                           23  13.9   M    2     41E.3               Elmenteita                      10-10.5                           23  11.3   N    2     45E.3.sup.CT               Elmenteita                      10   27  11.3   P    2     47E.3               Elmenteita                      10   27  11.3   O    2     51N.3               Nakuru 10-10.5                           29  40.1   P    2     52N.3               Nakuru 10-10.5                           29  40.1   P    2     42E.3               Elmenteita                      10-10.5                           23  13.9   N    2     50N.3               Nakuru 10-10.5                           29  40.1   N    2     Pseudomonas stutzeri.sup.T (NCIMB 11358)                                      --    --    wN2  Nakuru n.t. n.t.                               n.t.   A    --    Pseudomonas beijerinckii.sup.T (NCIMB 9041)                                      --    --    4E.1 Elmenteita                       9.5 35  n.t.   A    --    5E.1 Elmenteita                       9.5 35  n.t.   A    3     6B.1 Bogoria                      10.5 36  45     A    3     7B.1 Bogoria                      10.5 36  45     A    3     8B.1 Bogoria                      10.5 36  45     A    3     38E.2               Elmenteita                      n.t. n.t.                               n.t.   B    3     56E.4               Elmenteita                      10-10.5                           23  13.9   C    3     25B.1               Bogoria                      10.5 36  45     A    3     26N.1               Nakuru 10.5 36  30-45  A    3     11C.1               Crater  9.0 30  10     A    3     wB1  Bogoria                      n.t. n.t.                               n.t.   A    3     12C.1               Crater  9.0 30  10     A    3     28N.1.sup.CT               Nakuru 10.5 36  30-40  A    3     61N.4               Nakuru 10-10.5                           29  40.1   E    3     36E.3               Elmenteita                      n.t. n.t.                               n.t.   K    3     40E.3               Elmenteita                      10-10.5                           23  13.9   M    3     65B.4               Bogoria                      n.t. n.t.                               41.9   C    3     94LM.4               Little Magadi                      9-9.5                           81  35.0   L    3     19N.1               Nakuru 10.5 36  30-40  A    3     24B.1               Bogoria                      10.5 36  45     A    3     21M.1               Magadi 11.0 36  100    A    3     29C.1               Crater  9.0 30  10     A    3     35E.2               Elmenteita                      n.t. n.t.                               n.t.   I    3     37E.2               Elmenteita                      n.t. n.t.                               n.t.   J    3     48E.3               Elmenteita                      10.0 27  11.3   P    3     78LN.4               Little 8.5-9                           30   1.2   G               Naivasha    3     73aC.4               Crater n.t. n.t.                               10.2   D    3     75C.4               Crater n.t. n.t.                               n.t.   H    3     73bC.4               Crater n.t. n.t.                               10.2   D    3     74C.4               Crater n.t. n.t.                               10.2   H    3     77LN.4               Little 8.5-9                           30   1.2   F               Naivasha    3     wN1  Nakuru n.t. n.t.                               n.t.   A    3     49N.3               Nakuru 10-10.5                           29  40.1   Q    3     44E.3               Elmenteita                      10.0 27  13.9   O    3     58E.4               Elmenteita                      10.0 27  11.3   G    3     57E.4               Elmenteita                      10.0 27  11.3   C    4     wE5  Elmenteita                      n.t. n.t.                               n.t.   A    4     wB4.sup.CT               Bogoria                      n.t. n.t.                               n.t.   A    4     wNk1 Nakuru 10.5 n.t.                               19     A    4     wEl1 Elmenteita                      10.4 n.t.                               13     A    4     wEl2 Elmenteita                      10.4 n.t.                               13     A    5     9B.1 Bogoria                      10.5 36  45     A    5     16N.1               Nakuru 10.5 36  30-40  A    5     17N.1.sup.CT               Nakuru 10.5 36  30-40  A    5     22M.1               Magadi 11.0 36  100    A    6     18N.1               Nakuru 10.5 36  30-40  A    6     59E.4               Elmenteita                      10.0 31-33                               12.7   G    6     64B.4.sup.CT               Bogoria                      n.t. n.t.                               n.t.   E    6     63N.4               Nakuru  9.0 n.t.                               n.t.   C    6     53E.4               Elmenteita                      10-10.5                           23  13.9   G    --    92LM.4               Little Magadi                      9-9.5                           81  35.0   L    --    wBn5 Bogoria                      10.5 n.t.                               19     A    __________________________________________________________________________     *Clusters of microorganisms are obtained by analysis according to the     principles of numerical taxonomy using the S.sub.G /UPGMA method (see     discussion below and FIG. 1).     n.t. = not tested     The letter codes given for the Isolation Media refer to Appendix A.

Treatment of the Samples: Enrichment and Isolation of AlkaliphilicBacteria

A wide diversity of enrichment and isolation methods were applied. Someof the methods were specifically designed for the enrichment andisolation of alkaliphilic bacteria which exhibit specific types ofenzyme activity at an alkaline pH. Other techniques of a more generalnature were applied for the isolation of diverse sorts of alkaliphilicbacteria. In some cases, the specific conditions prevailing in the lakes(Table 2) were taken into account when experiments were performed forthe isolation of bacteria.

The different nutrient media employed for the isolation of the newalkaliphilic bacteria are designated Medium A - Medium Q. Thecomposition of the various media employed is shown in Appendix A.

For the isolation of non-specific alkaliphilic organotrophic bacteria,soda-lake water samples or dilutions thereof were streaked out on analkaline nutrient agar, pH 10-pH 10.5 (Medium A). Samples of a moresolid consistency, mud, sediment, etc. were first suspended in analkaline nutrient broth (Medium A) before spreading on an alkalinenutrient agar (Medium A). The bacteria were cultivated in a heatedincubator, preferably at 37° C. In some cases, the samples weresuspended in an alkaline nutrient broth (Medium A) and the bacteriacultivated by shaking, preferably at 37° C. for 2-3 days beforespreading the broth onto an alkaline nutrient agar (Medium A) for theisolation of bacterial colonies.

For the isolation of alkaliphilic bacteria exhibiting specific types ofenzyme activity, samples were spread onto alkaline nutrient agarcontaining specific substrates such as lactalbumin or casein or oliveoil. In some instances, the bacteria in the sample may be enriched for 1day or several weeks in a non-specific alkaline nutrient broth such asMedium A before spreading the broth onto an alkaline nutrient agarspecific for the detection of bacteria exhibiting enzyme activity suchas lipolytic or proteolytic activity.

Taxonomic Analysis

Seventy strains of bacteria isolated from in and around alkaline lakeswere assigned to the type of bacteria known as Gram-negative bacteria onthe basis of (1) the Dussault modification of the Gram's stainingreaction (Dussault, H. P., (1955), J. Bacteriol., 70, 484-485); (2) theKOH sensitivity test (Gregersen, T., (1978), Eur. J. Appl. Microbiol.and Biotech. 5, 123-127; Halebian, S. et al., (1981), J. Clin.Microbiol., 13, 444-448); (3) the aminopeptidase reaction (Cerny, G.,(1976), Eur. J. Appl. Microbiol., 3, 223-225; ibid, (1978), 5, 113-122);and in many cases, confirmation also on the basis of (4) a quinoneanalysis (Collins, M. D. & Jones, D., (1981), Microbiol. Rev., 45,316-354) using the method described by Collins, M. D. in ChemicalMethods in Bacterial Systematics (eds. M. Goodfellow & D. Minnikin) pp.267-288, Academic Press, London, 1985.

The seventy strains were tested for 104 characters. The results wereanalyzed using the principles of numerical taxonomy (Sneath, P. H. A.and Sokal, R. R., in Numerical Taxonomy, W. H. Freeman & Co., SanFrancisco, 1973). The characters tested and how they were tested arecompiled in Appendix B. In addition, Appendix C records how eachcharacter was coded for taxonomic analysis.

Since there are no well-documented strict or obligate non-phototrophic,alkaliphilic Gram-negative eubacteria known to the inventors, a diversecollection of 20 known Gram-negative bacteria were subjected as controlsto the same analysis, using modified pH conditions. These 20 knownreference bacteria are recorded in Table 4 from which it will be seenthat in most cases the "Type Strain" of the known species has been used.

                  TABLE 4    ______________________________________    Gram-Negative Non-Alkaliphilic Reference Strains    ______________________________________    (C.t.) Comamonas terrigena.sup.T NCIMB 8193    (P.p.) Pseudomonas putida.sup.T NCIMB 9494    (P.s.) Pseudomonas stutzeri.sup.T NCIMB 11358    (A.t.) "Alcaligenes tolerans" Leicester University strain    (V.c.) Vibrio costicola.sup.T NCIMB 701    (P.a.) Providencia alcalifaciens.sup.T NCTC 10286    (P.v.) Proteus vulgaris.sup.NT ATCC 13315    (M.v.) Moellerella wisconsensis.sup.T NCTC 12132    (E.t.) Edwardsiella tarda.sup.T NCTC 10396    (A.h.) Aeromonas hydrophila.sup.T NCTC 8049    (A.s.) Aeromonas sp S5 Leicester University strain    (F.a.) Flavobacterium aquatile.sup.T NCIMB 8694    (E.c.) Escherichia coli.sup.T NCTC 9001    (E.a.) Enterobacter aerogenes.sup.T NCTC 10006    (K.a.) Klebsiella pneumonia ATCC 15380 ("K. aerogenes")    (H.a.) Hafnia alvel.sup.T ATCC 13337    (C.f.) Citrobacter freundii.sup.T NCTC 9750    (S.m.) Serratia marcescens.sup.T NCTC 10211    (P.b.) Pseudomonas beijerinckii.sup.T NCIMB 9041    (H.e.) Halomonas elongata.sup.T ATCC 33173    ______________________________________     * abbreviation used in FIG. 1 and FIG. 2     .sup.T denotes "Type Strain"-     .sup.NT denotes "Neotype Strain"-

Analysis of Test Data

The Estimation of Taxonomic Resemblance

The phenetic data, consisting of 104 unit characters were scored asindicated in Appendix C, and set out in the form of an "n×t" matrix,whose t columns represent the t bacterial strains to be grouped on thebasis of resemblances, and whose n rows are the unit characters.Taxonomic resemblance of the bacterial strains was estimated by means ofa similarity coefficient (Sneath, P. H. A. and Sokal, R. R., NumericalTaxonomy, supra, pp. 114-187). Although many different coefficients havebeen used for biological classification, only a few have found regularuse in bacteriology. We have chosen to apply two associationcoefficients (Sneath, P. H. A. and Sokal, R. R., ibid, p. 129 et seq.),namely, the Gower and Jaccard coefficients. These have been frequentlyapplied to the analysis of bacteriological data and have a wideacceptance by those skilled in the art since they have been shown toresult in robust classifications.

The coded data were analyzed using the TAXPAK program package (Sackin,M. J., "Programmes for classification and identification". In Methods inMicrobiology, Volume 19 (eds. R. R. Colwell and R. Grigorova), pp.459-494, Academic Press, London, (1987)) run on a DEC VAX computer atthe University of Leicester, U.K.

A similarity matrix was constructed for all pairs of strains using theGower Coefficient (S_(G)) with the option of permitting negative matches(Sneath, P. H. A. and Sokal, R. R., supra, pp. 135-136) using theRTBNSIM program in TAXPAK. As the primary instrument of analysis and theone upon which most of the arguments presented herein are based, theGower Coefficient was chosen over other coefficients for generatingsimilarity matrices because it is applicable to all types of charactersor data, namely, two-state, multistate (ordered and qualitative), andquantitative.

Cluster analysis of the similarity matrix was accomplished using theUnweighted Pair Group Method with Arithmetic Averages (UPGMA) algorithm,also known as the Unweighted Average Linkage procedure, by running theSMATCLST sub-routine in TAXPAK.

The result of the cluster analysis is a dendrogram, a simplified versionof which is provided in FIG. 1. The dendrogram illustrates the levels ofsimilarity between the bacterial strains. The dendrogram is obtained byusing the DENDGR program in TAXPAK.

The phenetic data, omitting multistate characters (characters 1-5, 11and 12; Appendix C) and thus consisting of 193 unit characters, andscored in binary notation (positive=1, negative=0) were re-analyzedusing the Jaccard Coefficient (S_(J)) (Sneath, P. H. A. and Sokal, R.R., ibid, p. 131) by running the RTBNSIM program in TAXPAK. A furtherdendrogram was obtained by using the SMATCLST with UPGMA option andDENDGR sub-routines in TAXPAK. A simplified version of this dendrogramis illustrated in FIG. 2. Appendix E gives the percentage positivestates of characters in each cluster.

Results of the Cluster Analysis

S_(G) /UPGMA Method

FIG. 1 shows the results of cluster analysis, based on the GowerCoefficient and the UPGMA method, of 70 new, Gram-negative, alkaliphilicbacteria isolated from in and around alkaline lakes, together with 20known Gram-negative bacteria.

Six natural clusters or phenons of alkaliphilic bacteria which include65 of the 70 alkaliphilic strains are generated at the 73% similaritylevel. Although the choice of 73% for the level of delineation may seemarbitrary, it is in keeping with current practices in numerical taxonomy(Austin, B. and Priest, F., in Modern Bacterial Taxonomy, p. 37; VanNostrand Reinhold; Wokingham, U.K., (1986)). Placing the delineation ata lower percentage would combine groups of clearly unrelated organismswhile a higher percentage would produce a multitude of less well-definedclusters. At the 73% level, the individual clusters may representseparate bacterial genera. Furthermore, the significance of clusteringat this level is supported by chemotaxonomic data (see below) and thepattern of clusters obtained using the Jaccard Coefficient (FIG. 2).

The significance of the clustering at the 73% level is supported by theresults of the TESTDEN program. This program tests the significance ofall dichotomous pairs of clusters (comprising 4 or more strains) in aUPGMA generated dendrogram with squared Euclidean distances, or theircomplement, as a measurement. The program assumes that the clusters arehyperspherical. The critical overlap was set at 0.25%. As can be seenfrom Table 5, the separation of the clusters is highly significant.

                  TABLE 5    ______________________________________    Significance of the Clusters Generated by the S.sub.G /UPGMA    Method Provided by TESTDEN    CLUSTER separates from CLUSTER                          at Significance Level    ______________________________________    1                2        p = 0.99    1 + 2            3 + 4    p = 0.99    1 + 2 + 3 + 4    5 + 6    p < 0.90       1 + 2 + 3 + 4 + 5 + 6                     controls p = 0.99    5                6        p = 0.99    ______________________________________

A further measure of cluster separation can be estimated from theprobability of cluster overlap. This was achieved using the OVERMATprogram in TAXPAK with the critical overlap set out at 2.5%. As can beseen from Table 6, there is a greater than 95% probability of less than2.5% overlap between the clusters. For many of the cluster combinationsthe overlap is effectively nil. Only Clusters 3 and 4 have a lowerprobability of <2.5% overlap, but these clusters may be clearlydistinguished from one another on the basis of chemotaxonomic data (seebelow).

                  TABLE 6    ______________________________________    Percentage Probability that Cluster Overlap is <2.5%    CLUSTER 1        2       3      4     5     6    ______________________________________    2       95    3       99       95    4       95       95      90    5       99       >99     >99    >99    6       >99      >99     99     >99   >99    ______________________________________

The controls show that, as expected, the cluster analysis groups theEnterobacteriaceae separately. Additionally, the Aeromonas andPseudomonas species, included as controls, also group separately. Thisis entirely consistent with the current taxonomy of these organisms(Bergey's Manual of Systematic Bacteriology, volume 1, Williams andWilkins, Baltimore/London, 1984).

Five of the alkaliphilic strains fall outside the major clusters. Twostrains, 4E.1 and 5E.1 form a separate but related pair and areobviously associated with the major groups of alkaliphilic bacteria.Strain wN2 is also unclustered but is apparently related to aPseudomonas species and the major phenons of alkaliphilic bacteria.Strains 92LM.4 and wBn5 do not associate with the major alkaliphilicphenons and probably represent distinct groups of new alkaliphilicbacteria.

Clusters 1 and 2 are the only phenons which show an association withknown organisms, i.e. Pseudomonas and Comamonas species. The separationof Pseudomonas putida and Pseudomonas stutzeri into separate taxa isentirely in keeping with the current taxonomic status of these organisms(Palleroni, N. J. et al, (1973), Int. J. systematic Bacteriol., 23,333-339; Gavini, F. et al, (1989), ibid, 39, 135-144; Bergey's Manual ofSystematic Bacteriology, supra).

It was clear from the original dendrogram that Pseudomonas stutzeri isan outlier to Cluster 2 and is not closely related to the other membersof the cluster. This is seen when the Euclidean distances of the strainsfrom the centroid of the cluster are computed and used to calculate thecluster radius (Sneath, P. H. A. and Sokal, R. R., supra, pp. 194 etseq). The cluster radius is 3.91 (99% confidence level) and the meandistance of the strains from the centroid is 2.84 (standard deviation0.46). Pseudomonas stutzeri at a distance from the centroid of 3.91 isclearly at the very boundary of phenetic hyperspace which definesCluster 2.

A clear discrimination between Clusters 1 and 2 is possible using theconcept of the minimum discriminatory tests (see below).

Each of the alkaliphilic strains in Cluster 2 have been examined by twoindependent laboratories expert in the identification of bacteria,namely, the German Culture Collection (DSM, Braunschweig, FRG) and theLaboratory for Microbiology at Delft University of Technology, TheNetherlands. Neither of these laboratories was able to make a positiveidentification of the strains, although both agreed there was aresemblance with Pseudomonas either placing them in RNA homology group I(Palleroni, N. J. et al, supra) or more specifically in the Comamonastestosteroni/Pseudomonas alcaligenes or Pseudomonas pseudoalcaligenesgroups (Gavini, F. et al, supra). However, no Pseudomonas species areknown which are able to grow under the same highly alkaline conditions(pH 10) as the new strains described herein. An attempt was made tocultivate Pseudomonas pseudoalcaligenes^(T) DSM 50188 and Pseudomonasalcaligenes^(T) DSM 50342 in an alkaline broth medium (Medium A,Appendix A), but without success.

The results of these experts together with the discoveries describedhere, clearly indicate that these alkaliphilic strains in Clusters 1 and2 represent new species of bacteria.

Clusters 3, 4, 5 and 6 are discrete phenons distinguished from eachother on the basis of the minimum discriminatory tests (see below) andchemotaxonomic markers (see below). These phenons show no significantsimilarity with known groups of bacteria, and thus represent new generaor species.

Whole cell protein patterns generated by PAGE-electrophoresis indicatethat a number of strains are likely to be identical. Examples include:1E.1^(CT) and 2E.1; 6B.1, 7B.1 and 8B.1; 45E.3^(CT) and 47E.3. Thedendrogram reveals that these strains are related at an average S_(G)value of 92.3%, indicating a probable test error of 3.8% (Sheath, P. H.A. and Sokal, R. R., supra). Strains 73bC.4 and 74C.4, which appear tobe closely related (90% S_(G)), have similar but not identical gelpatterns.

S_(J) /UPGMA Method

The Jaccard coefficient is a useful adjunct to the Gower coefficient asit can be used to detect phenons in the latter generated by negativematches or distortions owing to undue weight being put on potentiallysubjective qualitative data. Consequently, the Jaccard coefficient isuseful for confirming the validity of clusters defined initially by theuse of the Gower coefficient. The Jaccard coefficient is particularlyuseful in comparing biochemically unreactive organisms (Austin, B., andPriest, F. G., supra, p. 37).

In the main, all of the clusters generated by the S_(G) /UPGMA methodare recovered in the dendrogram produced by the S_(J) /UPGMA method(FIG. 2). Although the composition of the clusters is virtuallyidentical in both dendrograms, a few strains have changed position.Non-clustering strains 4E.1 and 5E.1 move into Cluster 1/5, strains42E.3 and 50N.3 move from Cluster 2 to Cluster 3/4. The strains wNk2,Pseudomonas stutzeri, Pseudomonas putida, wE5 become nonclustering.

Not surprisingly, the S_(J) transformation combines (S_(G)) Clusters 1and 5. Both of these clusters are characterized as consisting ofbiochemically fairly unreactive strains. However, Clusters 1 and 5 areclearly distinct. Cluster 1 consists of strains producing cream/beige,circular colonies while the strains of Cluster 5 exclusively producebright yellow, irregular colonies.

Furthermore, the S_(J) transformation groups most of the strains ofCluster 4 with the strains of Cluster 3. However, it is evident from thechemotaxonomic data (see below), which shows that the strains of Cluster4 contain Q9 and the strains of Cluster 3 contain mainly Q6, that theseclusters should not be combined since they contain distinctly differentstrains. For these reasons, it is considered that the clusteringproduced by the S_(G) /UPGMA method is the better representation of theactual taxonomic status of these strains. However, the S_(J) /UPGMAserves to re-emphasize that with the single exception of a Comamonasspecies none of the known strains, not even the Pseudomonas controlstrains, bear significant resemblance to the clusters of the newalkaliphilic bacteria.

Chemotaxonomic Definition of the Clusters

Chemotaxonomy is the study of the chemical compositions of organisms inrelation to their systematics. The analysis of chromosomal DNA,ribosomal RNA, proteins, cell walls and membranes, for example, can givevaluable insights into taxonomic relationships and may be used as afurther tool to construct or to verify the taxonomies of microorganisms(Goodfellow, M. and Minnikin, D. E. in Chemical Methods in BacterialSystematics, (eds. Goodfellow, M. and Minnikin, D. E.), Academic Press,London and Orlando, Fla., (1985), pp. 1-15). However, it is not alwayspossible to decide a priori which type of chemical information will bemost diagnostic for a given classification. The amphipathic polarlipids, the major respiratory quinones, fatty acids located in thebacterial membranes and analysis of chromosomal DNA all have taxonomicsignificance for the classification of various bacteria (Lechevalier, H.and Lechevalier, M. P., in Microbial Lipids, volume 1 (eds. Ratledge, C.and Wilkinson, S. G.) Academic Press, London and San Diego, Calif.,(1988), pp. 869-902).

Polar Lipids

The extraction of polar lipids from bacteria and their analysis by twodimensional thin layer chromatography (2D-TLC) may yield patterns ofdiagnostic value. Stationary phase cells were extracted in 1:1 (v/v)CHCl₃ :CH₃ OH and examined by 2D-TLC as described by Ross, H. N. H.,Grant, W. D. and Harris, J. E., in Chemical Methods in BacterialSystematics, (eds. Goodfellow, M. and Minnikin, D. E.), Academic Press,London and Orlando, Fla. (1985), pp. 289-300. The types of lipidspresent on the chromatograms were visualized using a variety ofdifferential stains (Ross, H. N. M., et al., supra, p. 291; andTrincone, A., et al., J. Gen. Microbiol., (1990), 136, pp. 2327-2331).The identity of components were confirmed by co-chromatography withknown lipids.

The results of this analysis for representative strains of Gram-negativealkaliphiles are set out in Table 7. These show no clear polar lipidpattern which is distinct for any one cluster. All strains containphosphatidylglycerol, diphosphatidylglycerol, phosphatidylglycerolphosphate and phosphatidylethanolamine. In addition, certain strains,particularly in Cluster 3, contain phosphatidylglycerol sulphate (PGS).The distribution of PGS within Cluster 3 coincides broadly with thesuspected sub-group structure of the cluster evident from the pheneticand other chemotaxonamic data. PGS is therefore a non-exclusive markerfor Cluster 3.

We were surprised to find that a majority of the bacteria contained aglycolipid which on the basis of numerous co-chromatographic analysesappeared common to Gram-negative bacteria of the present invention.Glycolipids have not previously been demonstrated to be present inalkaliphilic bacteria (Krulwich, T. A., et al, CRC Critical Reviews inMicrobiology, (1988), 16, 15-36). Furthermore, at judged byco-chromatography of lipids obtained from several strains, theglycolipid is also found in Gram-positive alkaliphiles isolated fromsoda lakes. It is possible therefore, that the chemical structure of theglycolipid may be a chemotaxonomic marker for the obligate alkaliphiles.

                                      TABLE 7    __________________________________________________________________________    Polar Lipid Components of Gram-Negative Alkaliphilic Bacteria    CLUSTER          STRAIN PG DPG PGP                           PE PGS                                 GL AL UPL    __________________________________________________________________________          1E.1.sup.CT               + +  +   +          2E.1   +  +   +  ++    1     10B.1  +  +   +  +     3+          20N.1  +  +   +  ++          27M.1  +  +      ++          wNk.2  +  +   +  +           +          39E.3  +  +   +  +     +          41E.3  +  +   +  +     +          45E.3.sup.CT                 +  +   +  +     +    2     51N.3  +  +   +  +     +          52N.3  +  +   +  +     +          42E.3  +  +   +  +     +          50N.3  +  +   +  +  +          P.s.   +  +      +    --    5E.1   +  +   +  +  +  +     +          6B.1   +  +   +  +        +          7B.1   +  +   +  +     +          8B.1   +  +   +  +     +          25B.1  +  +   +  +     +          26N.1  +  +   +  +     +          12C.1  +  +   +  +     +  +    3     28N.1.sup.CT                 +  +   +  +  +          36E.2  +  +   +  +  +  +          40E.3  +  +   +  +  +  +          94LM.4 +  +   +  +  +  +          19N.1  +  +   +  +     +          24B.1  +  +   +  +     +          21M.1  +  +   +  +     +          29C.1  +  +   +  +  +          35E.2  +  +   +  +  +          37E.2  +  +   +  +  +  +          48E.3  +  +   +  +  +  +          73aC.4 +  +   +  +  +  +          74C.4  +  +   +  +  +  +          49E.3  +  +   +  +  +  +          44E.3  +  +   +  +  +  +          58E.4  +  +   +  +     +          wE5    +  +   +  +  +          wB4.sup.CT                 +  +   +  +    4     wNk1   +  +   +  +        +  +          wEl1   +  +   +  +        +  +          wE12   +  +   +  +           +          9B.1   +  +   +  +    5     16N.1  +  +   +  +          17N.1.sup.CT                 +  +   +  +     +          22M.1  +  +   +  +          18N.1  +  +   +  +     +          59E.4  +  +   +  +    6     64B.4.sup.CT                 +  +   +  +          63N.4  +  +   +  +     +          53E.4  +  +   +  +     +    __________________________________________________________________________     (PG) phosphatidylglycerol;     (DPG) diphosphatidylglycerol;     (PGP) phosphatidylglycerol phosphate;     (PE) phosphatidylethanolamine;     (PGS) phosphatidylglycerol sulphate;     (GL) unidentified glycolipid(s), α-naphthol positive (the number in     the column gives the number of positive spots on the TLC plate);     (AL) unidentified aminolipid (ninhydrin positive);     (UPL) unidentified phospholipid(s).

Isoprenoid Quinones

The isoprenoid or respiratory quinones are characteristic components ofthe plasma membrane of aerobic bacteria. There are two types;menaquinones and ubiquinones. The value of isoprenoid quinones astaxonomic criteria lies in the variation in the length of the polyprenylside-chain and the degree of saturation (Collins, M. D. and Jones, D.(1981), supra).

Freeze dried stationary phase bacterial cells were extracted, using amodified procedure of Collins, M. D. (in Chemical Methods in BacterialSystematics, supra, pp. 267-284), in 1:1 (v/v) CHCl₃ :CH₃ OH at 50° C.,for 16 hours. The quinones were examined by reverse phase thin layerchromatography as described by Collins, M. D. (supra).

The results of quinone analyses of nearly all the strains ofGram-negative alkaliphiles are illustrated in Table 8. All of thestrains tested contained exclusively ubiquinones which confirms theirstatus as Gram-negative bacteria (Collins, M. D. and Jones, D., supra).Table 8 shows quite clearly that the major ubiquinones are Q6 and Q9. Itis also evident that the strains containing Q6 are exclusive to cluster3 and that this distinguishes Cluster 3 from all the other clusterssince they contain strains possessing Q9 as the major ubiquinone.

                                      TABLE 8    __________________________________________________________________________    Major Respiratory Quinones of the Strains Arranged per Cluster    CLUSTER 1             CLUSTER 2                      CLUSTER 3                               CLUSTER 4                                        CLUSTER 5                                                 CLUSTER 6                                                         NON-CLUSTER    STRAIN         Q   STRAIN                  Q   STRAIN                           Q   STRAIN                                    Q   STRAIN                                             Q   STRAIN                                                      Q  STRAIN                                                               Q    __________________________________________________________________________    1E.1.sup.CT         Q9  39E.3                  Q9  6B.1 Q6  wE5  Q9  9B.1 Q9  18N.1                                                      Q9 wN.2  Q9    2E.1 Q9  41E.3                  Q9  7B.1 Q6, wB4.sup.CT                                    Q9  16N.1                                             Q9  59E.4                                                      Q9 5E.1  Q8                           Q9    wB2  Q9  45E.3.sup.CT                  Q9  8B.1 Q6, wNk1 Q9  17N.1.sup.CT                                             Q9, 64B.4.sup.CT                                                      Q9 92LM.4                                                               Q9                           Q9                Q10    wB5  Q9  47E.3                  Q9, 38E.2                           Q6  wEl1 Q9, 22M.1                                             Q9  63N.4                                                      Q9 wBn5  Q8,                  Q10               Q10(t)                     Q9(t)    wBs4 Q9  52N.3                  Q9  56E.4                           Q6  wEl2 Q9  22M.1                                             Q9  53E.4                                                      Q9    10B.1         Q9  42E.3                  Q9  25B.1                           Q6,                           Q9    20N.1         Q9  50N.3                  Q9  26N.1                           Q6    27M.1         Q9           12C.1                           Q6    C.t.*          Q8 +        28N.1.sup.CT                           Q6         Q9(t)!    wNk2 Q9           61N.4                           Q6    P.p.*          Q9!         36E.2                           Q6                      40E.3                           Q6,                           Q9                      65B.4                           Q6                      94LM.4                           Q6,                           Q9                      19N.1                           Q6,                           Q9                      24B.1                           Q6,                           Q9                      21M.1                           Q6                      29C.1                           Q6                      35E.2                           Q6                      37E.2                           Q6                      48E.3                           Q6                      78LN.4                           Q6                      73aC.4                           Q6                      75C.4                           Q6                      73bC.4                           Q6                      74C.4                           Q6,                           Q9                      77LN.4                           Q6                      49E.3                           Q6                      58E.4                           Q6                      57E.4                           Q6    __________________________________________________________________________     Q = Ubiquinone, the number indicates the number of sidechain isoprene     units.     (t) = trace     *C.t. = Comamonas terrigena.sup.T NCIMB 8193, the quinone result is     obtained from J. Tamaoka et al, International Journal of Systematic     Bacteriology, 37, 52-59, (1987).     P.p. = Pseudomonas putida.sup.T NCIMB 9494, the quinone result is obtaine     from M. D. Collins and D. Jones, Microbiological Reviews, 45, 316-354,     (1981).

Fatty Acids

The analysis of fatty acid profiles has had a significant impact onbacterial classification especially in the circumscription of genera andspecies among Gram-positive bacteria and actinomycetes (Kroppenstedt, R.M., in Chemical Methods in Bacterial Systematics (eds. M. Goodfellow andD. E. Minnikin), Academic Press; London and Orlando, Fla., (1985), pp.173-199); Lechevalier, H. and Lechevalier, M. P., supra.

Freeze dried stationary phase cells (200-300 mg) were extracted for 16hours at 75° C. in toluene:methanol:conc. sulphuric acid (2.5 ml:2.5ml:0.2 ml) and after cooling, the lipids were partitioned into hexane(twice times 1 ml). Residual acid was removed using NH₄ HCO₃. Lipidextracts were concentrated under O₂ -free N₂, dissolved in 300 μl hexaneand applied to preparative silica gel plates (Merck F254, Type T). Theplates were developed in hexane:diethyl ether 85:15 (v/v) and the fattyacid methyl esters scraped off, extracted with hexane and concentratedunder a stream of O₂ -free N₂.

The fatty acid methyl esters were dissolved in heptane and analyzed bygas chromatography using a Packard model 439 chromatograph equipped withflame ionization detectors. The samples were divided by a samplesplitter and analyzed simultaneously over two columns, namely, CP-SIL-88(Chrompack) (length 50 meter, internal diameter 0.22 mm) and Ultra-2(Hewlett/Packard) (length 50 m, internal diameter 0.20 mm). The carriergas was nitrogen; the injection temperature 120° C.; temperaturegradient 2.5° C. per minute to 240° C. and isothermal at 240° C. for 30minutes. Fatty acid methyl esters were assigned by reference to knownstandard mixtures. The identity of some peaks was confirmed by means ofgas chromatography-mass spectrometry using a Carlo Erba HRGC 5160 Megaseries gas chromatograph equipped with a CP-SIL-88 column (length 50meter, internal diameter 0.22 mm) with helium as carrier gas and directinjection into the source of a AMD 403 mass spectrometer.

The fatty acid compositions of representative individual Gram-negativebacteria are set out in Table 9. Table 10 shows the unique fatty acidprofiles of each of the clusters. Clusters 1, 2, 3 and 4 are fairlytypical of the majority of Gram-negative bacteria where the majorsaturated fatty acid is C16:0 with lesser amounts of C14:0 and C18:0.The major unsaturated fatty acids in these alkaliphilic bacteria areC16:0 and C18:1 (11-cis), which is also typical, as is the lack ofodd-numbered fatty acids (Wilkinson, S. G., in Microbial Lipids, volume1 (eds. Ratledge, C. and Wilkinson, S. G.), Academic Press, London andSan Diego, Calif., (1988), pp. 299-488). Minor amounts of C17:0 andC19:0 cyclopropane acids are found in some strains of Gram-negativebacteria. The strains of Cluster 3 exhibit fairly simple fatty acidprofiles with C16:0 and C18:1 contributing 67-88% of the total acids,and C16:1 plus C18:0 up to 20% of the remainder. Even so, the fatty acidpatterns support the notion that Cluster 3 contains several sub-groups,a conclusion that is also inferred from phenetic (numerical taxonomy)and polar lipid analyses (Table 7).

The strains of Cluster 1 can be distinguished from those of Cluster 2 onthe relative abundance of straight chain saturated and unsaturated fattyacids, as well as the percentage amounts of C18:1(11-cis). Thealkaliphilic bacteria of Clusters 1 and 2 have more complex fatty acidprofiles than those of Cluster 3, with many more minor components. Fromthe numerical taxonomy evidence, the alkaliphilic strains of Clusters 1and 2 exhibit some resemblance to Pseudomonas species. However, thetotal lack of any hydroxy-fatty acids which are typical of mostPseudomonas species, further indicating that a close relationship isdoubtful.

The strains of Clusters 5 and 6 are remarkable in that besidescontaining major amounts of C16:0, the other major fatty acids areodd-numbered branched chain acids (40-85%). Also, these strains lacksignificant amounts of C18:1 or any other unsaturated acids which arepresent in appreciable amounts in the alkaliphilic strains of Clusters1, 2, 3 and 4. The presence of large amounts of C15:0 and C17:0 iso andanteiso acids is characteristic of only a very few classes ofGram-negative bacteria, notably species from exotic environments such asThermus, or poorly defined taxa such as Flavobacterium (Wilkinson, S.G., supra). This result further emphasizes the novelty of thealkaliphilic strains of the present invention. The strains of Clusters 5and 6 can be distinguished from each other by the proportion ofbranched-chain fatty acids they contain and more especially by therelative proportions of even- and odd-numbered fatty acids.

    TABLE 9      - Fatty Acid Composition.sup.+      of Gram-Negative Alkaliphiles            CLUSTER → 1 2 3 4 5 6     non-clustering      FATTY ACID 1E.1.sup.CT 2E.1 45E.3.sup.CT 50E.3 25B.1 28N.1.sup.CT 36E.2 2     4B.1 37E.2 48E.3 WB4.sup.CT WEl1 9B.1 16N.1 17N.1.sup.CT 59E.4 64B.4.sup.     CT 5E.1 92LM.4      10:0      0.4     0.5      12:0 t t 3.6 4.7  0.5 0.5  0.3 t 0.9 0.2      12:1 0.2 0.3          0.8      13:0   <0.1      14:0 0.7 0.7 3.8 3.9 4.5 4.4 3.4 2.6 2.9 2.6 3.8 1.3 3.2 1.9 1.2 3.9     6.4 3.8 1.5      14:0 iso             0.7 t t 0.7 2.0      14:1 2.6 0.9    0.1      0.1      15:0  0.3 0.6 0.9 0.7 0.6   0.9 0.3 0.7 0.2    t 3.8 t      15:0 iso   0.1 0.2         8.7 9.3 8.2 3.9 12.6  44.0      15:0 anteiso   <0.1 0.3         32.3 27.1 35.3 24.8 18.1  9.9      15:0 cyclo           <0.1      16:0 29.0 32.0 28.5 34.4 37.3 24.1 42.0 36.7 33.3 26.0 26.8 26.9 22.5     17.4 12.5 26.3 40.5 74.7 18.7      16:0 iso  0.3  0.2         4.7 5.8 6.7 2.4 3.7  2.1      16:1 7.4 9.6 4.1 4.3 11.6 15.0 10.0 5.8 3.4 8.0 7.9 2.4      2.9             17:0 0.5 2.3 0.9 1.1  0.2   1.2 0.3 0.4 0.3    t 2.4      17:0 iso   0.2 0.6        0.3 3.2 6.0 5.1 0.6 1.7  15.7      17:0 anteiso             21.9 24.4 29.8 6.1 3.7  6.8      17:0 cyclo   0.4.sup.a 1.8     1.8 0.3 0.2      17:1 0.3 1.6   0.6      <0.1      17:1 br    1.7      18:0 12.0 4.7 17.9 10.8 8.9 0.2 0.9 2.0 0.6 1.1 5.2 3.5 2.3 5.3 1.2     16.2 4.8 2.6 1.5      18:0 unknown 0.2 0.4                5.9      18:1 9-cis 0.2 t 0.5 0.3  t t t 0.6 t t 2.5      18:1 9-trans 0.5 0.6 5.9 2.9 2.4 0.2 t 1.0 0.6 0.6 1.7 0.9 *0.5 *1.6     *1.0 *5.7 *0.5 *10.0      18:1 11-cis 42.0 44.0 23.9 24.1 27.5 54.2 43.0 50.6 41.6 57.7 47.4 48.1      18:1 unknown 0.2 0.3      18:2   0.5 1.9 2.7   0.5 t t 1.0 0.3    2.7      19:0   0.1      19:0 cyclo    1.3  0.4   12.9 2.0 1.0      19:1 br            11.2      20:0 0.4  5.3 2.7 2.4      1.3 0.4    4.6      20:1 3.6 1.2          0.3      22:0   3.3 1.5 1.4      0.8 0.2    2.6      24:0   0.2     * = includes all C18:1 isomers     .sup.+  = % total fatty acids     t = trace     br = branched     .sup.a  = C17:0 cyclo or C18:0 unknown

                                      TABLE 10    __________________________________________________________________________    Fatty Acid Profiles of the Clusters of Gram-Negative Alkaliphiles           Cluster           1      2      3A/B   3C     3D     4      5      6    __________________________________________________________________________    Predominant           C16:0  C16:0  C16:0  C16:0  C16:0  C16:0  C15:0 anteiso                                                            C15:0 br    Fatty Acids           C18:1 11-cis                  C18:0  C16:1  C18:1 11-cis                                       C18:1 11-cis                                              C18:1 11-cis                                                     C16:0  C16:0    (>10%)        C18:1 11-cis                         C18:1 11-cis                C17:0 anteiso    n-saturated           ˜40%                  60-65% 30-55% ˜40%                                       30-40% 30-40% 15-30% 55-60%    n-unsaturated           ˜60%                  ≈33%                         45-70% ˜60%                                       50-65% 50-60%  <2%   <10%    iso     <1%    <1%    0%     0%     0%     <1%   ˜20%                                                            10-20%    anteiso            0%     <1%    0%     0%     0%     0%    50-65% 20-30%    total   <1%    <3%    0%     0%     0%     1-12% >70%   ˜40%    branched    cyclo   0%     <5%           0%     2-15%  <2%    0%     0%    even carbon           >95%   >90%   >99%   >99%   >80%   >85%   20-35% 55-65%    no.    odd carbon            <5%   <10%    <1%    <1%   <20%   <15%   65-80% 35-45%    no.    additional    C17:0 cyclo          C17:0 cyclo    markers       C19:0 cyclo          C19:0 cyclo                  C17:1 br    __________________________________________________________________________     br = branched

Nucleic Acids

An essential component of any taxonomic study is an analysis of thegenetic material--the nucleic acids. The composition of chromosomal DNAis unaffected by the growth conditions of the organism and anappropriate analysis may confirm or refute the taxonomic position of theorganism. Chromosomal DNA may be analyzed by the determination of thebase composition (G+C mol %) of individual strains, and the basesequence homologies between pairs of strains by DNA-DNA reassociation(hybridization) (Owen, R. J. and Pitcher, D., in Chemical Methods inBacterial Systematics (eds. M. Goodfellow and D. E. Minnikin), AcademicPress, London and Orlando, Fla. (1985), pp. 67-93).

DNA Base Composition

The guanine plus cytosine (G+C mol %) composition is constant for thechromosomal DNA from any given organism. Closely related organisms havesimilar G+C compositions. However, G+C results must be interpretedwithin the context of independent taxonomic data since similar G+C mol %of DNA samples from different organisms does not in itself implybiological relatedness.

DNA was extracted from cells grown to exponential phase in Medium A bythe chloroform:phenol method and was precipitated with ethanol. Basecomposition was determined by the thermal denaturation method (Marmur,J. and Doty, P. (1962), J. Mol. Biol., 3, 585-594) on a Phillips modelPV8764 spectrophotometer with temperature programming. A second methodinvolved HPLC analysis on a Beckman system gold using a Beckmanultrasphere ODS column and 0.04M potassium dihydrogen phosphate plusacetonitrile (9+1, v/v) as eluent at a flow rate of 1.5 ml/min, aftertreatment of the DNA with nuclease P1 and alkaline phosphatase.

The results of these analyses are set out in Table 11.The G+C mol %values for the alkaliphilic bacteria cover a range of 30 mol %(37.6-67.1 mol %). However, within the clusters the variation is only3-7 mol %, which further confirms that the strains within a cluster areclosely related to each other.

                  TABLE 11    ______________________________________    DNA Base Composition of Gram-Negative    Alkaliphilic Bacteria                      G + C mol %    Cluster     Strain      HPLC    T.sub.M    ______________________________________    1           2E.1        55.2                wBs4                51.2                20N.1       51.1                wNk2                53.0    2           42E.2       62.7    3           wB1                 63.0                28N.1.sup.CT                            64.1                37E.2       67.1                wN1                 64.8    4           wE5                 58.5                wB4.sup.CT          65.3                wNk1                61.0                wE11                59.7                wE12                58.1    5           17N.1.sup.CT                            50.0                22M.1       43.8    6           64B.4.sup.CT                            41.0                53E.4       37.6    non         wN2                 64.1                wBn5                54.6    ______________________________________

DNA-DNA Molecular Hybridization

The method used was essentially that of Crosa, J. H. et al. (Int. J.Systematic Bacteriol., 29, 328-332, 1979). Tritium labelled DNA wasprepared using a nick-translation kit (Amersham, N5000) according to themanufacturer's instructions. The reassociation mixtures were incubatedat 65° C. for 16 hours. The results are set out in Table 12 from whichit can be seen that the DNA sequence homology is higher within theclusters that between the clusters.

                                      TABLE 12    __________________________________________________________________________    Inter-Cluster and Intra-Cluster DNA-DNA Homology    Values for Gram-Negative Alkaliphilic Bacteria              Cluster              1   2    3    4   5    6    Cluster         Strain              2E.1                  45E.3.sup.CT                       28N.1.sup.CT                            wE12                                17N.1.sup.CT                                     64B.4.sup.CT    __________________________________________________________________________    1    2E.1 100       33   35  25   25         20N.1               56    2    45E.3.sup.CT                  100        25         42E.3     76            30    3    28N.1.sup.CT               20  34  100   26  30   30         56E.4          51              21M.1              53         37E.2     37         44E.3          55    4    wE12               100         wB4.sup.CT     41   43    5    17N.1.sup.CT               45  44   36   24 100   44         22M.1                   65    6    64B.4.sup.CT               21  31   35   25  34  100         53E.4                        60         SS    17                20   20    __________________________________________________________________________     The values give the percent hybridization between the strains (rows) and     H.sup.3 -labelled strains (columns).     SS = salmon sperm DNA.

Determination of Representative Strains

The centroid of each individual cluster generated by the S_(G) /UPGMAmethod was computed using the RGROUPS program in TAXPAK. The centroid ofa cluster of points representing real organisms projected intohyperspace represents a hypothetical average organism. The centroidrarely, if ever represents a real organism. Therefore, the Euclideandistances of each of the members of the cluster from the centroid of thecluster were calculated in order to establish which strain was closestto the hypothetical average organism. The strain closest to the centroidwas designated the "centrotype" organism (indicated with the superscript"CT").

The centrotype organism can be thought of as the "Type Strain" whichmost closely represents the essential and discriminating features ofeach particular cluster. The centrotype strains are recorded in Table13.

                  TABLE 13    ______________________________________    Centrotype Strains    Num-       Mean Euclidean  Centrotype           ber of  Distance of             Euclidean           Strains Strains                 Distance    Cluster           in      from      Standard      from    Number Cluster Centroid  Deviation                                     Strain                                           Centroid    ______________________________________    1      11      3.67      0.30    1E.1  2.88    2       9      3.20      0.52    45E.3 2.30    3      34      3.52      0.32    28N.1 2.90    4       5      3.97      0.29    wB4   2.87    5       4      3.25      0.29    17N.1 2.13    6       5      3.11      0.41    64B.4 1.93    ______________________________________

A description of each of the centrotype organisms has been made so as tobe able to distinguish these organisms from all other bacteriapreviously known and described. In addition, the minimum number ofdiscriminating tests to define each cluster has been computed so that itmay be clearly seen that the clusters containing these novel bacteriacan be easily distinguished from each other and from all other knownbacteria.

Description of Centrotype Strains

Strain 1E.1^(CT) (Cluster 1)

An aerobic, motile, Gram-negative rod-shaped bacterium, 1·7-3·3μm×0·5-0·7 μm.

Obligate alkaliphile, grows best between pH 9 and pH 10.

On alkaline-agar, (Medium A) forms smooth, cream colored colonies,initially translucent but becoming opaque after a few days. The coloniesare circular, entire and convex, 2-3 mm in diameter.

In alkaline-broth, (Medium A) growth (37° C.) is flocculent with theformation of a sediment and surface pellicle.

Grows well between 20° C. and 40° C. Grows slowly at 10°-15° C. Nogrowth at 8° C. or 45° C.

    ______________________________________    KOH test:         positive    Aminopeptidase:   weak positive    Oxidase:          negative    Catalase:         positive    NaCl tolerance:   0% to <8%. No growth at 8%    Hydrolysis of Gelatin:                      positive    Hydrolysis of Starch:                      positive    Major polar lipid components:                      phosphatidylglycerol                      diphosphatidylglycerol                      phosphatidylglycerol phosphate                      phosphatidylethanolamine    Major ubiquinone: Q9    Major fatty acids:                      C16:0, C18:0, 11-cis C18:1    ______________________________________

Chemoorganotroph. Grows on complex substrates such as yeast extract andpeptones. Growth on simple sugars and organic acids very restricted(e.g., growth only observed on ribose, sucrose and pyruvate).

Strain 45E.3^(CT) (Cluster 2)

An aerobic, Gram-negative, rod-shaped bacterium, 3-4.5 μm×0.6 μm. Motileby a single polar flagellum.

Obligate alkaliphile growing between pH 7.8 and pH 11.2. Onalkaline-agar, (Medium A) forms smooth, opaque, cream colored colonies,1-2 mm in diameter. The colonies are circular, convex and entire.

In alkaline-broth, (Medium A) growth (37° C.) is slow, slight with aneven turbidity, surface pellicle and no sediment.

Grows well between 20° C. and 40° C. Grows slowly at 10° C. No growth at8° C. or 45° C.

    ______________________________________    KOH test:         positive    Aminopeptidase:   positive    Oxidase:          positive    Catalase:         positive    NaCl tolerance:   0% to 12%. Growth at 12%                      is slow                      No growth at 15%    Hydrolysis of Gelatin:                      positive    Hydrolysis of Starch:                      positive (weak)    Major polar lipid components:                      phosphatidylglycerol                      diphosphatidylglycerol                      phosphatidylglycerol phosphate                      phosphatidylethanolamine                      glycolipid (α-naphthol positive)    Major ubiquinone: Q9    Major fatty acids:                      C16:0, C18:0, 11-cis C18:1    ______________________________________

Chemoorganotroph. Grows on complex substrates such as yeast extract andpeptones. No growth on simple sugars. Grows on organic acids (e.g.,fumarate, succinate, pyruvate, acetate, lactate) and some fatty acids(e.g., propionate, valerate) and amino acids (e.g., proline, alanins,phenylalanine).

Strain 28N.1^(CT) (Cluster 3)

An aerobic, motile, Gram-negative, rod-shaped bacterium, 4·8-5·5μm×0·6-0·8 μm. Obligate alkaliphile growing between pH 8·5 and pH 10·7.

On alkaline-agar, (Medium A) forms smooth, circular, opaque colonieswith a stringy texture. The colonies have a convex elevation and entiremargin. The colony color is initially cream/beige becoming pink after afew days.

In alkaline-broth, (Medium A) growth (37° C.) is heavy, flocculent witha surface pellicle and a sediment.

Grows well between 20° C. and 45° C. Grows slowly at 10° C. and 15° C.No growth at 50° C.

    ______________________________________    KOH test:         positive    Aminopeptidase:   positive    Oxidase:          positive    Catalase:         positive    NaCl tolerance:   0% to 12%. No growth at 15%    Hydrolysis of Gelatin:                      negative    Hydrolysis of Starch:                      positive    Major polar lipid components:                      phosphatidylglycerol                      diphosphatidylglycerol                      phosphatidylglycerol phosphate                      phosphatidylglycerol sulphate                      phosphatidylethanolamine    Major ubiquinone: Q6    Major fatty acids:                      C16:0, C16:1, 11-cis C18:1    G + C:            64.1 mol % (HPLC)    ______________________________________

Chemoorganotroph. Grows well on complex substrates such as yeast extractand peptones. Grows on simple sugars, organic acids, fatty acids andamino acids.

Strain wB4^(CT) (cluster 4)

An aerobic, Gram-negative, rod-shaped bacterium, 3-4 μm×0.6-0.8 μm,frequently occurring as pairs of cells.

Alkaliphile, grows well between pH 7.5 and pH 10.9.

On alkaline-agar, (Medium A) forms smooth, beige to brown colonies. Thecolonies are somewhat variable: 1 to >5 mm in size, circular toirregular in form, low convex or raised in elevation with an undulate orentire margin.

In alkaline broth, (Medium A) growth (37° C.) is flocculent, sedimentforming with a surface pellicle.

Grows best between 15° C. and 45° C., no growth at 50° C.

    ______________________________________    KOH test:        positive    Aminopeptidase:  positive    Oxidase:         very weakly positive, may be                     seen as negative    Catalase:        positive    NaCl tolerance:  0% to ≧12%, no growth at 15%    Hydrolysis of Gelatin:                     negative    Hydrolysis of Starch:                     negative    Major polar lipid components:                     phosphatidylglycerol                     diphosphatidylglycerol                     phosphatidylglycerol phosphate                     phosphatidylethanolamine    Major ubiquinone:                     Q9    Major fatty acids:                     C16:0, 11-cis C18:1    G + C:           65.3 mol % (T.sub.M)    ______________________________________

Chemoorganotroph. Grows well on complex substrates such as yeastextract. Growth on simple sugars is restricted. Grows on organic acids(e.g., lactate, acetate, fumarate), fatty acids (e.g., propionate,valerate, caprate) and amino acids (e.g., proline, serine, lysine).

Strain 17N.1^(CT) (cluster 5)

An aerobic, Gram-negative, long, thin, rod-shaped, bacterium, 5.5-10.5μm×0.6 μm, sometimes forming short chains of cells. With agepleomorphic, peculiar swollen forms predominate.

Obligate alkaliphile, grows best between pH 8 and pH 10.5.

On alkaline-agar, (Medium A) forms smooth, opaque, yellow, colonies, 2-3mm in diameter. The colonies vary from circular to irregular in form,with a convex to umbonate elevation, and entire, undulate or lobatemargin, depending upon age.

In alkaline-broth (Medium A), growth (37° C.) is even and sedimentforming with no surface pellicle.

Grows well between 15° C. and 37° C. Grows slowly at 10° C. and not atall at 8° C. No growth at 40° C. or above.

    ______________________________________    KOH test:        positive    Aminopeptidase:  weakly positive    Oxidase:         negative    Catalase:        positive    NaCl tolerance:  0% to <12%, grows best at 0%                     NaCl    Hydrolysis of Gelatin:                     positive    Hydrolysis of Starch:                     weakly positive    Major polar lipid components:                     phosphatidylglycerol                     diphosphatidylglycerol                     phosphatidylglycerol phosphate                     phosphatidylethanolamine                     glycolipid (α-naphthol positive)    Major ubiquinone:                     Q9, Q10    Major fatty acids:                     C15:0 anteiso, C16:0,                     C17:0 anteiso    G + C            :50.0 mol % (HPLC)    ______________________________________

Chemoorganotroph. Grows well on complex substrates such as yeastextract. Growth on simple sugars is restricted (e.g., growth observedonly on fructose; no growth observed on glucose, ribose, lactose). Growson organic acids (e.g., fumarate, succinate, pyruvate, 2-ketogluconate)and amino acids.

Strain 64B.4^(CT) (cluster 6)

An aerobic, Gram-negative, rod-shaped bacterium 2.0-3.5 μm×0.8-1.0 μm.

Obligate alkaliphile, grows best between pH 8.2 and pH 10.9.

On alkaline-agar, (Medium A) forms smooth, opaque colonies, first creamyyellow in color, becoming beige with age. The colonies are about 4 mm indiameter, circular becoming irregular; flat or low convex in elevationbecoming convex; with an entire margin becoming undulate.

In alkaline-broth (Medium A), growth (37° C.) is even, sediment formingwith no surface pellicle.

Grows well at 15° C. to 45° C., no growth at 10° C. or 50° C.

    ______________________________________    KOH test:        positive    Aminopeptidase:  negative    Oxidase:         positive    Catalase:        positive    NaCl tolerance:  0% to ≦12%, no growth at 15%    Hydrolysis of Gelatin:                     positive    Hydrolysis of Starch:                     weakly positive    Major polar lipid components:                     phosphatidylglycerol                     diphosphatidylglycerol                     phosphatidylglycerol phosphate                     phosphatidylethanolamine                     glycolipid (α-naphthol positive)    Major ubiquinone:                     Q9    Major fatty acids:                     C15:0 iso, C15:0 anteiso,                     C16:0 G + C: 41.0 ± 0.9 mol %                     (HPLC)    ______________________________________

Chemoorganotroph. Grows well on complex substances such as yeastextract. Grows on some simple sugars (e.g., glucose, ribose, maltose andfructose), organic acids (e.g., acetate, lactate, citrate and fumarate),some fatty acids (e.g., propionate and caprate) and amino acids (e.g.,proline, histidine and alanine).

Non-Clustering Strains

The strains which do not fall into the clusters defined here are alsonovel bacteria not previously known or described. These strains, codedwN2, 4E.1, 5E.1, 92LM.4 and wBn5, may represent rarer varieties ofalkaliphilic bacteria and are probably members of clusters of bacteriarepresenting new genera and species at present not described. Adescription of these "non-clustering" strains has been made so as to beable to distinguish these organisms from all other bacteria previouslyknown and described.

Strain wN2

An aerobic, Gram-negative, motile, rod-shaped bacterium, frequently inpairs.

Obligate alkaliphile, grows best between pH 9 and pH 10.

On alkaline-agar, (Medium A) forms smooth, translucent, beige coloredcolonies, 1-2 mm in diameter. The colonies are circular, convex with anentire margin.

In alkaline-broth (Medium A), growth (37° C.) is flocculent with a ringor surface pellicle and formation of a sediment.

Grows well at 20° C. to 30° C. No growth at 15° C. or 40° C.

    ______________________________________    KOH test:     positive    Aminopeptidase:                  weak positive    Oxidase:      weak positive    Catalase:     positive    NaCl tolerance:                  obligate halophile, growth at 4% NaCl                  no growth at 0% or 8% NaCl    Hydrolysis of Gelatin:                  slow positive    Hydrolysis of Starch:                  positive    Major ubiquinone:                  Q9    G + C:        64.1 (T.sub.M)    ______________________________________

Chemoorganotroph. Metabolically unreactive. No growth on simple sugarsor organic acids. Grows on complex substrates such as yeast extract andpeptones, and on some amino acids.

Strain 4E.1

An aerobic, Gram-negative, motile, rod-shaped bacterium, 1.7-5.2 μm×0.75μm.

Obligate alkaliphile, grows best between pH 8.2 and pH 10.9.

On alkaline-agar, (Medium A) forms smooth, opaque, beige or browncolored colonies, 2-4 mm in diameter. The colonies are circular in form,convex in elevation, with an entire margin.

In alkaline-broth (Medium A), growth (37° C.) is heavy and flocculentwith a sediment and surface pellicle.

Grows well between 20° C. and 37° C. Grows very slowly at 10° C. and notat all at 8° C. No growth at 40° C. or above.

    ______________________________________    KOH test:    positive    Aminopeptidase:                 positive    Oxidase:     very weakly positive, can appear negative    Catalase:    positive    NaCl tolerance:                 0% to 12%, may grow weakly at 15%                 no growth at 20%    Hydrolysis of Gelatin:                 negative    Hydrolysis of Starch:                 negative    ______________________________________

Chemoorganotroph. Does not grow on simple sugars, except for ribose.Grows well on complex substrates such as yeast extract, and on organicacids (e.g., succinate, pyruvate, citrate, malonate, acetate andlactate), fatty acids (e.g., propionate, valerate and suberate), andamino acids (e.g., proline, serine, histidine and lysine).

Strain 5E.1

An aerobic, Gram-negative, rod-shaped bacterium, 3.0-5.3 μm×1.3 μm.

Obligate alkaliphile, grows best between pH 9 and pH 10.5.

On alkaline-agar, (Medium A) forms smooth, opaque, brown coloredcolonies, 3-4 mm in diameter. The colonies are fairly irregular in form,generally flat to slightly umbonate in elevation with a lobate margin.

In alkaline-broth (Medium A), growth (37° C.) is moderate to heavy,becoming flocculent with a sediment and surface pellicle.

Grows well between 20° C. and 40° C. Grows slowly at 10° C. No growth at45° C.

    ______________________________________    KOH test:         positive    Aminopeptidase:   positive    Oxidase:          negative    Catalase:         positive    NaCl tolerance:   0% to 12%,                      may grow weakly at 15%                      no growth at 20%    Hydrolysis of Gelatin:                      positive    Hydrolysis of Starch:                      weakly positive    Major polar lipid components:                      phosphatidylglycerol                      diphosphatidylglycerol                      phosphatidylglycerol phosphate                      phosphatidylglycerol sulphate                      phosphatidylethanolamine                      glycolipid (α-naphthol positive)    Major ubiquinone: Q8    Major fatty acids:                      C16:0, C18:1    ______________________________________

Chemoorganotroph. Does not grow on simple sugars. Grows well on complexsubstrates such as yeast extract, organic acids (e.g., pyruvate,citrate, acetate and lactate), fatty acids (e.g., propionate, caprateand valerate) and amino acids (e.g., proline, alanine and lysine).

Strain 92LM.4

An aerobic, Gram-negative, rod-shaped bacterium, 2.0-3.5 μm×0.5-1.0 μm.

Obligate alkaliphile, no growth below pH 7.5.

On alkaline-agar, (Medium A) forms smooth, cream colored colonies,initially translucent but becoming opaque. The colonies develop fromcircular, entire to irregular, lobate in form, with a convex elevation.

In alkaline-broth (Medium A), growth (37° C.) is slow, slight,flocculent with a sediment but no surface pellicle.

Grows between 10° C. and 40° C., no growth at 8° C. or 45° C.

    ______________________________________    KOH test:      positive    Aminopeptidase:                   negative    Oxidase:       positive    Catalase:      positive    NaCl tolerance:                   0% to 15%, growth at 15% is slow                   no growth at 20%    Hydrolysis of Gelatin:                   positive    Hydrolysis of Starch:                   weakly positive    Major ubiquinone:                   Q9    Major fatty acids:                   C15:0 iso, C15:0 anteiso,                   C16:0, C17:0 iso    ______________________________________

Chemoorganotroph. Grows on complex substrates such as yeast extract andpeptones, and a variety of sugars, organic acids and amino acids.

Strain wBn5

An aerobic, Gram-negative, small, rod-shaped bacterium, frequentlyforming short chains of cells.

Obligate alkaliphile, no growth below pH 8.

On alkaline-agar, (Medium A) forms smooth, circular, convex colonieswith an entire margin, about 1 mm in diameter. The colonies areinitially cream/beige, transparent becoming opaque, brown.

In alkaline-broth (Medium A), growth (37° C.) is initially evenly turbidwith a sediment but no surface pellicle becoming after 4 days flocculentwith formation of a pellicle.

Grows at 30° C. and 37° C. No growth at 40° C.

    ______________________________________    KOH test:     positive    Aminopeptidase:                  positive    Oxidase:      positive    Catalase:     positive    NaCl tolerance:                  obligate halophile. Growth at 4% NaCl                  no growth at 0% or 8%    Hydrolysis of Gelatin:                  slow positive    Hydrolysis of Starch:                  negative    Major ubiquinone:                  Q8, Q9    G + C:        54.6 mol % (T.sub.M)    ______________________________________

Chemoorganotroph. Grows on a range of complex substrates such as yeastextract and peptones, as well as sugars, organic acids, fatty acids andamino acids.

Cluster Definition by the Calculation of the Minimum Number ofDiscriminatory Tests, and the Construction of a Probability Matrix forthe Identification of Gram-Negative Alkaliphiles

One of the purposes of a numerical classification study is to use thephenetic data, which defines the clusters at a selected similaritylevel, for the assignment or identification of unknown strains. Theclassification test data can be used to determine the minimum set oftests which are required to define the clusters at the 73% (S_(G))similarity level, and to identify those characters which are mostdiagnostic (predictive) for the individual clusters. In other words, theminimum number of tests required to assign an unknown organism to apre-determined cluster with a high degree of predictability.

From the minimum discriminatory tests, a probability matrix can beconstructed for the identification of unknown strains. The analysis isachieved by using a combination of the CHARSEP and DIACHAR (TAXPAK) andMCHOICE (not on TAXPAK but available by Data-Mail from the University ofLeicester, U.K.) programs. An evaluation of the identification matrix isprovided by using the MOSTTYP and OVERMAT programs. Practical examplesof the use of these programs for the probabilistic identification ofbacteria have been published by Williams, S. T., et al., (1983), J. Gen.Microbiol., 129, pp. 1815-1830; and Priest, F. G. and Alexander, B.,(1988), J. Gen. Microbiol., 134, pp. 3011-3018; ibid, (1990), 136, pp.367-376.

A "n×t" table was constructed from the test data using characters 6 to10 and 13 to 104 (Appendix C) scored in binary notation (positive=1,negative=0). This data matrix was supplemented with the following fourextra character states:

105! Bright yellow colonies (character number 1, Appendix C)

106! Translucent colonies (grown on Medium A, Appendix A)

107! Lipase (lipolytic activity on olive oil (Medium M))

108! Oxidass positive within 10 secs. (test 9, Appendix B)

The data matrix is first examined using the CHARSEP program whichcalculates separation indices and thus the diagnostic value of theindividual characters for discriminating between the clusters. Testswith a VSP index>25% (Sneath, P. H. A., (1979), Computers andGeosciences, 5, 349-357) are accepted, characters with a low diagnosticvalue (VSP<25%) were rejected. A preference is made for characters withthe highest VSP indices, provided that the criteria in the DIACHAR andMCHOICE programs are also met. In this example, 38 tests have a VSPindex>25%, and 9 of the 24 characters finally chosen have a VSPindex>50% (Table 11).

The data matrix is next re-examined by means of the DIACHAR program,which determines the most diagnostic character states of each of theclusters. The number of character states was set at 10. This resultallows the choice of mutually exclusive character states between theclusters. As many of these tests as possible are retained in the finalidentification matrix of minimum discriminatory tests; in this examplebetween 6 and 9 diagnostic characters per cluster. The remaining, unusedtests are also noted and may be applied as additional tests for theconfirmation of identification (Table 12).

The MCHOICE program ranks the tests in groups which can be displayed inthe form of a dendrogram using the MDEND subroutine. The groups identifytests with similar discriminatory value, thus allowing the rejection oftests which fail to make a significant discrimination as well asallowing choices to be made between tests of equal or very similardiagnostic value.

Table 13 shows the set of 24 tests which is the minimum number requiredto define the clusters and which can be used for the assignment ofunknown strains. In addition, Table 13 shows the identification matrixwhich consists of the percentage of positive characters which define theclusters on the basis of the 24 minimum discriminatory tests. This iscomputed by the IDMAT program.

                  TABLE 14    ______________________________________    Separation Values of Characters used for the    Minimum Discriminatory Tests    CHARACTER          VSP Index    ______________________________________      23!   N-acetylglucosamine                           35.4      26!   Saccharose     44.8      27!   Maltose        41.4      32!   Lactate        51.6      41!   Propionate     60.9      43!   Valerate       63.4      44!   Citrate        45.1      45!   Histidine      38.0      47!   Glycogen       31.7      51!   3-hydroxybutyrate                           66.1      52!   4-hydroxybanzoate                           38.0      58!   Leucine arylamidase                           36.6      59!   Valine arylamidase                           50.5      64!   Phosphohydrolase                           52.8      65!   α-galactosidase                           33.9      85!   Ampicillin     36.8      92!   Fusidic Acid   68.7      93!   Methicillin    58.3      99!   Polymixin      62.8     102!   Vancomycin     48.3    ______________________________________

                  TABLE 15    ______________________________________    Discriminatory Tests for Each of the Six Clusters (S.sub.G)    Positive         Negative    ______________________________________    Cluster 1:    cream, circular, opaque, mucoid colonies.      58!         Leucine arylamidase                           23!  N-acetylglucosamide (9%)         (91%)             27!  Maltose (9%)      59!         Valine arylamidase                           41!  Propionate (9%)         (91%)             42!  Caprate      64!         Phosphohydrolase (91%)                           43!  Valerate (9%)      99!         Polymixin (89%)   ∴!                                Citrate (9%)                           45!  Histidine                           47!  Glycogen (9%)                           52!  4-hydroxybenzoate                           65!  α-galactosidase    Cluster 2:    small, cream, translucent colonies.      21!         Starch            23!  N-acetylglucosamine      31!         Acetate           26!  Saccharose      41!         Propionate        45!  Histidine      43!         Valearate         50!  2-ketogluconate      53!         Proline           52!  4-hydroxybenzoate     107!         Lipase            68!  α-glucosidase     108!         Oxidase (within 10 secs.)                           69!  β-glucosidase                           92!  Fusidic Acid    Cluster 3:    cream, opaque colonies.      31!         Acetate           64!  Phosphohydrolase (3%)      32!         Lactate           65!  α-galactosidase (3%)      41!         Propionate (94%)                           92!  Fusidic Acid (3%)      43!         Valerate (97%)    96!  Tetracycline (3%)      44!         Citrate (94%)    102!  Vancomycin      51!         3-hydroxybutyrate                          104!  Bacitracin         (94%)      53!         Proline      58!         Leucine arylamidase         (94%)    Cluster 4:    beige to brown, opaque colonies.      32!         Lactate           45!  Histidine      33!         Alanine           85!  Ampicillin      48!         3-hydroxybutyrate                           86!  Naladixic acid      59!         Valine arylamidase                           88!  Trimethoprim      99!         Polymixin         89!  Penicillin G                           93!  Methicillin    Cluster 5:    bright yellow  105!, opaque colonies.      64!         Phosphohydrolase                           23!  N-acetylglucosamine      65!         α-galactosidase                           32!  Lactate      66!         β-galactosidase                           33!  L-alanine      85!         Ampicillin        34!  Mannitol      92!         Fusidic Acid      41!  Propionate      93!         Methicillin       42!  Caprate      96!         Tetracyclinee     43!  Valerate     102!         Vancomycin        45!  Histidine     104!         Bactracin         48!  3-hydroxybenzoate                           51!  3-hydroxybutyrate                           52!  4-hydroxybenzoate                           99!  Polymixin    Cluster 6:    cream, irregular, flat colonies.      21!         starch            17!  Pyruvate      23!         N-acetylglucosamine                           52!  4-hydroxybenzoate      26!         Saccharose        58!  Leucine arylamidase      27!         Maltose           59!  Valine arylamidase      31!         Acetate           65!  α-galactosidase      33!         Alanine           99!  Polymixin      44!         Citrate      47!         Glycogen      51!         3-hydroxybutyrate      89!         Penicillin G      92!         Fusidic Acid      93!         Methicillin      96!         Tetracyclinee     104!         Bacitracin    ______________________________________     Note: The numbers in square brackets proceeding the character state refer     to the character states and unit tests in Appendices B and C. The     percentage in parenthesis refers to positive character states.

                  TABLE 16    ______________________________________    A Probability Matrix for the Identification of    Alkaliphiles: Percentage Distribution of Positive    Discriminatory Characters Which Define the    Clusters of Gram-Negative Alkaliphilic    Bacteria at the 73% Level (S.sub.G)                  CLUSTER    TEST            1     2      3    4    5    6    ______________________________________      23!         N-acetylglucosamine                        13     0    26   20   0   100      26!         Saccharose     25     0    74   20   25  100      27!         Maltose        25     0    68   60   50  100      32!         Lactate        38     50  100  100   0    40      41!         Propionate      0    100   91   60   0    80      43!         Valerate       13    100   97   80   0    40      44!         Citrate        13     50   94   20   50  100      45!         Histidine       0     0    71   0    0    80      47!         Glycogen        0     13   26   20   25  100      51!         3-hydroxybutyrate                        13     25   94  100   0   100      52!         4-hydroxybenzoate                         0     0    71   80   0    0      58!         Leucine arylamidase                        88     63   94   60   50   0      59!         Valine arylamidase                        88     25   65  100   25   0      64!         Phosphohydrolase                        88     13   3    20   75   40      65!         α-galactosidase                         0     0    3    20   75   0      85!         Ampicillin     50     63   56   0   100   80      92!         Fusidic Acid   25     0    3    20  100  100      93!         Methicillin    50     13   50   0   100  100      99!         Polymixin      88     50   81  100   0    0     102!         Vancomycin     13     13   3    20  100   75     105!         Yellow colony   0     0    0    0   100   0     106!         Translucent colony                         0    100   3    0    0    0     107!         Lipase          0    100   21   0    0    0     108!         Oxidase (10 secs)                        25     88   6    0    0    0    ______________________________________

Evaluation of the Discriminatory Tests and Assessment of the Reliabilityof Identification

The evaluation of the discriminatory tests has two aspects. Firstly, thevalidity of the tests can be analyzed using practical examples, whichcan be further evaluated using statistical theory, or the tests can bedirectly subjected to theoretical assessment using statistical methods.

ILLUSTRATION 1 A Practical Evaluation of the Discriminatory Tests

Many workers assess the accuracy of the discriminatory tests only byredetermining the character states of selected cluster representatives.This approach has been used here for the centrotype strains (see below).A far more stringent approach which is seldom applied, is to examine allthe strains which were used in the original numerical taxonomicanalysis. When subjected to cluster analysis using only the dataacquired from the derived set of minimum discriminatory tests, thereconstructed dendrogram can be compared with the original. Using onlythe 24 discriminatory tests previously described (Table 16), the data(two-state, binary form) for all 70 of the novel Gram-negativealkaliphilic bacteria were subjected to cluster analysis by the S_(G)/UPGMA method. The reconstructed dendrogram is reproduced in FIG. 3.This reconstructed dendrogram compares very favorably with the originaldendrogram (FIG. 1).

Although there has been some rearrangement of position of the clusters,their composition is largely unchanged and they are defined atapproximately the same similarity level as the original. Cluster 4however, has combined with Cluster 3, with a single strain moving toCluster 1. This further serves to emphasize the difficulty of definingCluster 4 on phenetic data alone. It has been stressed several timesthat supplementary chemotaxonomic data are required to make the properdistinction between Cluster 3 and Cluster 4.

In both the original dendrogram and the reconstruction (FIG. 3), Cluster3 appears to comprise several subclusters above the 73% similaritylevel. The fine structure of cluster 3 is also supported by thechemotaxonomic data (see above).

ILLUSTRATION 2 A Theoretical Evaluation of the Discriminatory Tests

An assessment of cluster overlap is achieved using the OVERMAT program.This program examines the matrix constructed from the percentagepositive values for the selected character states against a criticaloverlap value by considering the clusters defined by the coordinates ofthe centroid and the cluster radius (twice root mean square of thedistances of the strains from the centroid). If there is significantoverlap between the clusters, unknown strains may not identify withsufficient confidence to any one of them (Sneath, P. H. A. and Sokal, R.R., supra, p. 394-400). At a chosen critical overlap value of 2.5%(which is a more stringent condition than is used by most workers: seePriest, F. G. and Alexander, B., (1988), supra; and Williams, S. T. etal., (1983), supra) there was no significant overlap between theclusters (95% confidence level) except between Cluster 3 and Cluster 4where the actual overlap was calculated to be 4%. However,chemotaxonomic data (see above) was not taken into account whenconstructing the identification matrix. On the basis of quinoneanalyses, strains from Cluster 3 can be distinguished from the strainsof Cluster 4.

ILLUSTRATION 3 A Theoretical Assessment of the Reliability ofIdentification

The hypothetical median organism (HMO) is another estimate of the"average" organism in a cluster (Sneath, P. H. A. and Sokal, R. R.,supra, pp. 194 et seq.). A HMO is not a real strain but a hypotheticalorganism possessing the most common state for each character. TheMOSTTYP program calculates HMO's for each cluster in the identificationmatrix and then attempts to identify them. In other words, MOSTTYP is aprogram to evaluate an identification matrix by calculatingidentification scores of the most typical strains against the clusters.A good identification matrix should give a high probability of a HMObeing reassigned to its own cluster. The results of this analysis werevery satisfactory (Table 17), especially since MOSTTYP was programed toconsider only the first 20 diagnostic tests of the identification matrix(Table 16), i.e. excluding tests 105-108. Each HMO was reassigned to itsoriginal cluster with Willcox probabilities of 0.998-1.000 (Willcox, W.R. et al., (1973) J. Gen. Microbiol., 77, 317-330). The TaxonomicDistances were all low and the standard errors of the Taxonomic Distancewere all negative, indicating that the HMO's were all closer to thecentroid of the cluster than the average for the cluster (Table 17).

                  TABLE 17    ______________________________________    Identification Scores for the Hypothetical Median Organism    of each cluster provided by the MOSTTYP Program           Identification Score             Willcox    Taxonomic  Standard Error of    CLUSTER  Probability                        Distance   Taxonomic Distance    ______________________________________    1        0.999      0.194      -2.742    2        0.999      0.236      -2.214    3        1.000      0.231      -2.115    4        0.998      0.195      -2.998    5        1.000      0.217      -1.839    6        1.000      0.182      -2.502    ______________________________________

ILLUSTRATION 4 A Practical Evaluation of Identification Score

Identification of strains using the minimum set of discriminatory testsis achieved using the MATIDEN program in TAXPAK. The program comparespresence-absence data for an unknown strain against each cluster in turnin an identification matrix of percentage positive characters.Identification coefficients are computed, namely Willcox probability,Taxonomic Distance and the Standard Error of the Taxonomic Distance. Theresults are displayed, showing the identification scores to the bestcluster and to the two next best alternative clusters. Additionally, theatypical results ("characters against") are recorded. In an analysisusing data from real strains, the centrotypes were reassigned to theiroriginal clusters with Willcox probabilities of 0.9996-1.000 (Table 18).The Taxonomic Distances were low. The Standard Errors of the TaxonomicDistance were all negative indicating that the centrotypes were closerto the centroid of the cluster than the average for the cluster.

                                      TABLE 18    __________________________________________________________________________    Identification Scores for the Centrotype Organisms of    Each Cluster Provided by thye MATIDEN Program                        Identification Score                        Willcox                              Taxonomic                                     Standard    Cluster         Strain              Assigned to Cluster                        Probability                              Distance (D)                                     Error of D    __________________________________________________________________________    1    2E.1 1         1.000 0.309  -0.283    2    45E.3.sup.CT              2         1.000 0.226  -1.749    3    28N.1.sup.CT              3         1.000 0.305  -0.622    4    wB4.sup.CT              4         0.9996                              0.265  -1.092    5    17N.1.sup.CT              5         0.9999                              0.255  -0.478    6    64N.4.sup.CT              6         1.000 0.211  -1.126    __________________________________________________________________________

ILLUSTRATION 5 Identification of Unknown Isolates

The identification matrix was assessed for the ability to assign unknownGram-negative alkaliphiles to the clusters defined herein. The criteriafor a successful identification were:

(a) bacteria isolated from a habitat similar to, but geographicallyseparate from, the East African soda lakes;

(b) a Willcox probability greater than 0.95 and low values for TaxonomicDistance and its standard error (<3);

(c) an identification score to the best cluster significantly betterthan those against the two next best alternatives;

(d) "characters against" the best cluster should be zero or few innumber.

Unknown microorganisms may be examined using the minimum tests listed inTable 16. The character states are determined and identification scoresobtained using the MATIDEN program. This program compares the characterstates of the unknown with the identification matrix determined for allof the predetermined clusters, computes the best match and assigns theunknown to the most appropriate cluster.

A Willcox probability is calculated to determine the acceptability ofidentification. Willcox probabilities of 0.85 and 0.95 have beenaccepted as criteria for a successful identification (Williams, S. T.,et al. (1983), supra; Priest, F. G. and Alexander, B., (1988), supra).The Taxonomic Distance of the unknown from the cluster centroid iscalculated and may be compared to the radius of the cluster. TheStandard Error of the Taxonomic Distance should be less than the uppervalue of +3.0 suggested by Sneath, P. H. A. ((1979), pp. 195-213).Moreover, physical characteristics, additional biochemical data andchemotaxomomic markers may be used to further confirm the identity ofthe unknown in a particular cluster.

The results provided by these five illustrations, together with thestatistical data provided by the numerical taxonomic analysis and thechemotaxonomic data, indicate a robust classification which identifies 6major groups of new, Gram-negative, alkaliphilic bacteria.

Phylogenetic Analysis

Phylogenetics is the study of relationships based on ancestry, in otherwords phylogeny reflects the evolutionary pathways of organisms (Austin,B. and Priest, F. G., (1986), pp. 5-9, 57-60, 74-81, ibid). The currentpractice in microbiology is to use macromolecule sequences in theconstruction of cladograms (genealogical trees) (Woese, C. R. (1987),Microbiol. Rev. 51, 221-271) which reveal evolutionary relationships.The most useful molecules for revealing prokaryotic phylogeny are theribosomal RNAs (Woese, C. R. (1985), ibid, pp. 227 et seq.).

Chromosomal DNA was extracted using the method of Sambrook, J. et al.,(1989), (in Molecular Cloning, a laboratory manual, (Sambrook, J.,Fritsch, E. F. and Maniatis, T., eds.) Cold Spring Harbor LaboratoryPress, N.Y.). The gene encoding 16S rRNA was amplified by the polymerasechain reaction (PCR) using modifications of the method of Weisburg, W.G. et al., (1991), (J. Bacteriol. 173, 697-703,). Twenty-five cycles ofdenaturation (94° C. for 1 minute), annealing (55° C. for 1 minute) andextension (72° C. for 1 minute) were performed. DNA was denatured at 98°C. for 5 minutes prior to adding the enzyme, and at the end of the lastcycle, the extension time was lengthened to 7 minutes. The primers usedare written in 5' to 3' orientation and the numbering based on the 16SrRNA sequence of E.coli (Brosius, J. et al., (1978), Proc. Natl. Acad.Sci. (USA), 75, 4801-4805).

The forward amplification primer was fD1, AGAGTTTGATCCTGGCTCAG (position8-27) and the reverse primer was a mixture of rP1,ACGGTTACCTTGTTACGACTT; rP2, ACGGCTACCTTGTTACGACTT and rP3,ACGGATACCTTGTTACGACTT (position 1512-1492).

The amplified PCR product was purified using QIAGEN purification system(Qiagen, Inc., Chatsworth, Calif.). Direct automated sequencing of 16SrDNA amplified by PCR was performed on a model 373A DNA Sequencer™(Applied Biosystems, Foster City, Calif.) using a modification of themethod of Hiraishi, A. (1992), (Lett. Appl. Microbiol. 15, 210-213,).Sequencing was performed by dye-labelled dideoxy-sequencing using themethod of Johnson-Dow, L. et al., (1987), (Biotechniques, 5, 754-765)with 25 asymmetrical thermal cycles.

A mixture of 3 reverse primers and 3 forward primers was used:

    ______________________________________    rD2;   GAATTACCGCGGCGGCTG (position 536-519),           GAATTACCGCGGCTGCTG (position 536-519),           GTATTACCGCGGCGGCTG (position 536-519),           GTATTACCGCGGCTGCTG (position 536-519);    rD3;   CCGTCAATTCCTTTGAGTTT (position 926-907),           CCGTCAATTCCTTTAAGTTT (position 926-907),           CCGTCAATTCATTTGAGTTT (position 926-907),           CCGTCAATTCATTTAAGTTT (position 926-907);    rD4;   ACGGGCGGTGTGTGTAC (position 1406-1390),           ACGGGCGGTGTGTGTGC (position 1406-1390);    fD2;   CAGCCGCCGCGGTAATTC (position 519-536),           CAGCAGCCGCGGTAATTC (position 519-536),           CAGCCGCCGCGGTAAATC (position 519-536),           CAGCAGCCGCGGTAAATC (position 519-536);    fD3;   AAACTTAAATGAATTGACGG (position 907-926),           AAACTCAAATGAATTGACGG (position 907-926),           AAACTTAAAGGAATTGACGG (position 907-926),           AAACTCAAAGGAATTGACGG (position 907-926);    fD4;   GTACACACACCGCCCGT (position 1390-1406),           GCACACACACCGCCCGT (position 1390-1406).    ______________________________________

The strains used in the sequence analysis are listed in Table 19.

                  TABLE 19    ______________________________________    Source of 16S rRNA Sequences for Phylogenetic Analysis                 Access   Strain    Species      Number   Specification                                     Reference    ______________________________________    Magatibacter          1E.1.sup.CT                                     This study    afermentans    Sodabacter            28N.1.sup.CT                                     This study    nakuruai    Igatibacter           64B.4.sup.CT                                     This studt    hanningtonii    Halomonas    X67023   ATCC 33173 Gauthier, M.J.,    elongata                         et al. (1992)                                     (Int. J. Syst.                                     Bacteriol. 42,                                     568-576).    Halomonas    M59153   ATCC 19717 Woese, C.R.    halmophila                       (unpublished)    Pseudomonas  X06684   DSM 50071  Toschka, H.Y.,    aeruginosa                       et al. (1988)                                     (NuCl. Acids                                     Res. 16,                                     2348-2348).    Marinomonas  X67023   ATCC 27119 Gauthier, M.J.,    vaga                             et al. (1992)                                     ibid.    Legionella   M36024   NCTC 11286 Fry, N.K.,    pneumophila                      et al. (1991)                                     (J. Gen.                                     Microbiol. 137,                                     1215-1222).    Acinetobacter                 M34139   ATCC 33604 Woese, C.R.    calcoaceticus                    (unpublished)    Alteromonas  X67024   ATCC 14393 Gauthier, M.J.,    haloplanktis                     et al. (1992)                                     ibid.    Aeromonas sp.                 X60417   ATCC 39541 Martinez-                                     Murcia, A.J.,                                     et al. (1992)                                     (Int. J. Syst.                                     Bacteriol. 42,                                     412-421).    Plesiomonas  X60418   NCIMB 9242 Martinez-    shigelloides                     Murcia, A.J.,                                     et al. (1992)                                     ibid.    Proteus      X07652   IFAM 1731  Stackebrandt,    vulgaris                         E. (unpub-                                     lished)    E. coli      M24828              Carbon, P.,                                     et al. (1979)                                     (Eur. J.                                     Biochem. 100,                                     399-410).    Marinobacter X67022   ATCC 49840 Gauthier, M.J.,    hydrocarbonoclasticus            et al. (1992)                                     ibid.    Oceanospirillum                 M22365   ATCC 11338 Woese, C.R.    linum                            (Unpublished)    ______________________________________

Sequences were accessed from GenBank and EMBL databases or from theRibosomal Database Project (RDP) (Olsen, G. J., et al., (1992), NucleicAcids Research, supplement, 20, 2199-2200,). A preliminary alignment ofsequences was performed using the program Clustal V (Higgens, D. G., etal., CABIOS, 8, 189-191,) and refined by employing secondary structurecriteria of the RDP. The aligned sequences were subjected tophylogenetic analysis using programs in versions 3.4 or 3.5c of thePHYLIP package (Felsenstein, J. (1989), Cladistics, 5, 164-166).Evolutionary distances were calculated using the nucleotide substitutionmodel of Jukes, T. H. and Cantor, R. R. (1969) (in Mammalian proteinmetabolism, (Munzo, H. N., ed.), Academic Press, New York, pp. 21-132)in the program DNADIST. A phylogenetic tree (FIG. 4) based on thesevalues was constructed using the least squares algorithm of Fitch, W. M.and Margoliash, E. (1967), (Science, 155, 279-284) in the program FITCH.

The sequence of the 16S rRNA gene from strain 1E.1^(CT) consisted of1450 bases, (96% complete); strain 28N.1^(CT) consisted of 1494 bases,(98% complete); and strain 64B.4^(CT) consisted of 1461 bases, (95%complete). The phylogenetic analysis revealed that all 3 alkaliphilicstrains of the present invention belong to the gamma-3 subdivision ofthe Proteobacteria (Woese, C. R., et al, (1985) (Syst. Appl. Microbiol.6, 25-33). Strains 28N.1^(CT) and 64B.4^(CT) are evidently more closelyrelated to each other than to strain 1E.1^(CT). Strain 1E.1^(CT) clearlyrepresents a new genus within the Enteric-Aeromonas-Vibrio branch of thegamma-3 subdivision of the Proteobacteria for which we give the nameMagatibacter (gen. nov.). Therefore, strain 1E.1^(CT) which we nameMagatibacter afermentans (gen. nov., sp. nov.) is the Type Strainrepresenting all the strains of cluster 1. Strains 28N.1^(CT) and64B.4^(CT) are clearly members of the Halomonas group, which includesDeleya, a deep branch of the gamma-3 subdivision of the Proteobacteria(Woese, C. R., et al, (1985), ibid). The data (summarized in Table 20)indicates that both strain 28N.1^(CT) and 64B.4^(CT) deserve separatetaxonomic status at the genus level. Although the percentagedissimilarity between strain 64B.4^(CT), Halomonas elonagata ATCC 33173and Halomonas halmophilum ATCC 19717 (formally Flavobacteriumhalmophilum) based on the analysis of the 16S rRNA gene sequences israther small, 4.9% and 4.8% respectively, the significantly lower G+Ccontent of strain 64B.4^(CT) (41 mol %) compared to the 59-63 mol %range for Halomonas (Vreeland, R. H. (1992), in The Prokaryotes, 2nded., vol. 4, (Balows, A., Truper, H. G., Dworkin, M., Harder, W. andSchleifer, K-H., eds.), pp. 3181-3188, Springer-Verlag, New York)indicates a fundamental difference. Since strain 28N.1^(CT) is no closerrelated to strain 64B.4^(CT) than 64B.4^(CT) is related to Halomonas(Table 20A) and 28N.1^(CT) is more distantly related to Halomonas than64B.4^(CT), a separate generic status for the 2 obligately alkaliphilicstrains of the present invention is undoubtedly indicated. Thisconclusion is further supported by G+C content, DNA-DNA reassociationstudies and chemotaxonomic evidence (Table 20). Conclusive evidence forthe separation of strain 28N.1^(CT) and strain 64B.4^(CT) from Halomonasas separate genera is the lack of 3 (strain 28N.1^(CT)) or 4 (strain64B.4^(CT)) of the 6 unique signature sequences which are common toHalomonas (Table 20G) (Woese, C. R. et al., (1985), ibid).

A distinction from the genus Deleya is more difficult to assess sinceonly partial 16S rRNA gene sequences are available for D. marina(Kita-Tsukanoto, K., et al., (1993), (Int. J. Syst. Bacteriol. 43,8-19). In order to minimize possible errors that might be introduced bycomparison of partial sequence data, only complete 16S rRNA genesequences were used in the construction of the phylogenetic tree (FIG.4). However, when the partial 16S rRNA sequence of Deleya marina(Kita-Tsukanoto, K., et al., (1993), ibid) was aligned with sequencesfrom Halomonas elongata, Halomonas halmophila and strain 64B.4^(CT), andthe percentage dissimilarity between species calculated (Table 21) aclear distinction was revealed. All this evidence, together with theunique alkaliphilic phenotype of the strains of the present inventionindicates that strain 28N.1^(CT) and strain 64B.4^(CT) represent new,independent genera. Therefore, strain 28N.1^(CT) which we nameSodabacter nakuruai (gen. nov., sp. nov.) is the Type Strainrepresenting all the strains of cluster 3, and strain 64B.4^(CT) whichwe name Igatibacter hanningtonii (gen. nov., sp. nov.) is the TypeStrain representing all strains of cluster 6. From the precedingevidence and that of the numerical taxonomy and chemotaxonomy, it may besafely concluded that all the alkaliphilic bacteria of the presentinvention represent species of new genera.

                  TABLE 20    ______________________________________    Comparison of Halomonas with Alkaliphilic Strain 28N.1    and 64B.4.    TABLE 20A.    Relative Phylogenetic Distance             H.h.       H.e    64B.4    H.h    H.e    64B.4    54         66    28N.1    71         75     54    ______________________________________    TABLE 20B.    G + C content mol %           Halomonas                   59-63           28N.1   64           64B.4   41    ______________________________________    TABLE 20C.    DNA-DNA Hybridization                     28    Strain           N.1    64B.4    28N.1            100     30    64B.4             35    100    ______________________________________    TABLE 20D.    Major Respiratory Quinone           Halomonas                   Q9           28N.1   Q6           64B.4   Q9    ______________________________________    TABLE 20E.    Major Fatty Acids    Halomonas         C12:0, 3-OH-C12:0,                      iso-C15:0, C16:0,                      C16:1, C17:0,                      9-trans-C18:1    28N.1             C16:0, C16:1,                      11-cis-C18:1    64B.4             iso-C15:0,                      anteiso-C15:0                      C16:0    ______________________________________    TABLE 20F.    Polar Lipid Composition    Halomonas        PG DPG    28N.1            PG DPG PGP PE PGS    64B.4            PG DPG PGP PE    ______________________________________    TABLE 20G.    RNA Signature Sequences              Halo-              monas   28N.1        64B.4    CCUAACUUCG              +       -            -                      CUAACCUUCG   CCUAACCUUCN    UUAAUACCCG              +       +            -                                   UUAAUACCCU    AUAACUUG  +       -            -                      AUAACCUG     UNANCGUG    CCCUCG    +       -            -                      CCUUCG       CCUUCG    UCUCAG    +       +            +    UUAACG    +       +            +    ______________________________________

                  TABLE 21    ______________________________________    Percent Dissimilarity                       Deleya  Halomonas             64B.4     marina  elongata    ______________________________________    64B.4      --    Deleya     6.5         --    marina    Halomonas  5.9         8.0     --    elongata    Halomonas                      2.9    halmophilum    ______________________________________

Production and Application of Alkali-Tolerant Enzymes

The alkaliphilic microorganisms of the present invention produce avariety of alkali-tolerant enzymes. Examples of enzyme activitiespresent in representative strains of the Gram-negative bacteria of thepresent invention may be found in Appendices D and E. These enzymes arecapable of performing their functions at an extremely high pH, makingthem uniquely suited for their application in a variety of processesrequiring such enzymatic activity in high pH environments or reactionconditions.

Examples of the various applications for alkali-tolerant enzymes are indetergent compositions, leather tanning, food treatment, waste treatmentand in the textile industry. These enzymes may also be used forbiotransformations, especially in the preparation of pure enantiomers.

The alkaliphilic bacteria of the present invention may easily bescreened for the production of alkali-tolerant lipases, proteases andstarch-degrading enzymes, inter alia, using the methods describedherein.

The broth in which alkaliphilic bacteria are cultured typically containsone or more types of enzymatic activity. The broth containing the enzymeor enzymes may be used directly in the desired process after the removalof the bacteria therefrom by means of centrifugation or filtration, forexample.

If desired, the culture filtrate may be concentrated by freeze drying,before or after dialysis, or by ultrafiltration. The enzymes may also berecovered by precipitation and filtration. Alternatively, the enzyme orenzymes contained in the broth may be isolated and purified bychromatographic means or by gel electrophoresis, for example, beforebeing applied to the desired process. The exact methods used to treatthe culture filtrate and/or to extract and/or purify the alkali-tolerantenzymes is not critical to the present invention, and may be determinedby one skilled in the art.

The genes encoding alkali-tolerant enzymes of interest may be cloned andexpressed in organisms capable of expressing the desired enzyme in apure or easily recoverable form.

The following examples are provided to illustrate methods for theidentification of Gram-negative alkaliphilic bacteria of the presentinvention, as well as methods of screening these alkaliphilic bacteriafor the presence of various alkali-tolerant enzymes and methods for thesubsequent production and application of these enzymes in industrialprocesses. These examples are not to be construed so as to limit thescope of the present invention.

EXAMPLE 1 Identification of Unknown Isolates

six strains of Gram-negative, alkaliphilic bacteria were isolated fromMono Lake, a hypersaline, alkaline lake situated in California, U.S.A.(Javor, B., in Hypersaline Environments, Springer Verlag, Berlin andHeidelberg (1988), pp. 303-305). The strains were isolated from samplesof partially submerged soda-encrusted wood, tufa and soda-soil collectedfrom the environs of Mono Lake (Calif., U.S.A.) in May, 1990 byenrichment culture at 37° C. in Medium A (Appendix A). The six strainsare described in Table 22. The strains were examined using 21 of the 24minimum tests listed in Table 16. The character states were determinedand identification scores obtained using the MATIDEN program. Theresults are outlined in Table 23.

                  TABLE 22    ______________________________________    Alkaliphilic Strains from Mono Lake            Colony               Cell    Strain          Sample  Color   Form   Elevation                                         Margin                                               Shape    ______________________________________    ML005 tufa    beige   circular                                 convex  entire                                               rod    ML104 wood    pink/   circular                                 convex  entire                                               rod                  beige    ML201 wood    yellow  circular                                 convex  entire                                               rod    ML203 wood    pink/   circular                                 convex  entire                                               rod                  beige    ML206 wood    yellow  circular                                 convex  entire                                               short                                               rod    ML301 soil    beige   circular                                 convex  entire                                               rod    ______________________________________

                  TABLE 23    ______________________________________    Example of the Output from the MATIDEN Program to Identify    Six Unknown Strains against the Identification Matrix    ______________________________________    A. Reference Number of unknown is ML005                       Percent in:                    Value in           Next    Character       unknown  Best Taxon                                       Best Taxon    ______________________________________     23! N-acetylglucosamine                        +        26      20     26! Saccharose     +        74      20     27! Maltose        +        68      60     32! Lactate        +        99      99     41! Propionate     +        91      60     43! Valerate       +        97      80     44! Citrate        +        94      20     45! Histidine      +        71      1     47! Glycogen       +        26      20     51! 3-Hydroxybutyrate                        +        94      99     52! 4-Hydroxybenzoate                        -        71      80     58! Leucine arylamidase                        +        94      60     59! Valine arylamidase                        +        65      99     64! Phosphohydrolase                        -        3       20     65! α-Galactosidase                        n.t.     3       20     85! Ampicillin     -        56      1     92! Fusidic Acid   -        3       20     93! Methicillin    -        50      1     99! Polymix        +        81      99     102!         Vancomycin     -        3       20     105!         Yellow colony  -        1       1     106!         Translucent colony                        n.t.     3       1     107!         Lipase         n.t.     21      1     108!         Oxidase (10 sec.)                        +        6       1    ______________________________________    n.t. = not tested    Isolate ML005 best identification is Cluster 3. Scores    for coefficients:1 (Willcox probability), 2 (Taxonomic Dis-    tance), 3 (Standard Error of Taxonomic Distance).              1              2      3    ______________________________________    Cluster 3 0.9999         0.407  1.475    Cluster 4 0.8266 × 10.sup.-4                             0.526  4.590    Cluster 2 0.4086 × 10.sup.-7                             0.584  6.296    ______________________________________                 Cluster 3    Characters against                   % in Taxon                             Value in unknown    ______________________________________     108! Oxidase (10 sec.)                   6         +    ______________________________________    Additional characters that assist in separating                  Cluster 3          Cluster 4                  %           from   %    ______________________________________     106! Translucent colony                   3                 99     107! Lipase  21                 99    ______________________________________    B. Reference Number of unknown is ML104                       Percent in:                    Value in           Next    Character       unknown  Best Taxon                                       Best Taxon    ______________________________________     23! N-acetylglucosamine                        -        13      1     26! Saccharose     -        25      1     27! Maltose        -        25      1     32! Lactate        -        38      50     41! Propionate     -        1       99     43! Valerate       -        13      99     44! Citrate        -        13      50     45! Histidine      -        1       1     47! Glycogen       -        1       13     51! 3-Hydroxybutyrate                        -        13      25     52! 4-Hydroxybenzoate                        -        1       1     58! Leucine arylamidase                        +        88      63     59! Valine arylamidase                        +        88      25     64! Phosphohydrolase                        +        88      13     65! α-Galactosidase                        n.t.     1       1     85! Ampicillin     -        50      63     92! Fusidic Acid   -        25      1     93! Methicillin    -        50      13     99! Polymix        +        88      50     102!         Vancomycin     -        13      13     105!         Yellow colony  -        1       1     106!         Translucent colony                        n.t.     1       99     107!         Lipase         n.t.     1       99     108!         Oxidase (10 sec.)                        +        25      88    ______________________________________    n.t. = not tested    Isolate ML104 best identification is Cluster 1. Scores    for coefficients: 1 (Willcox probability), 2 (Taxonomic Dis-    tance), 3 (Standard Error of Taxonomic Distance).              1              2      3    ______________________________________    Cluster 1 0.9999         0.271  -1.108    Cluster 2 0.825 × 10.sup.-5                             0.473  3.797    Cluster 4 0.407 × 10.sup.-7                             0.534  4.767    ______________________________________                 Cluster 1    Characters against                   % in Taxon                             Value in unknown    ______________________________________    (none)    ______________________________________    Additional characters that assist in separating                  Cluster 1          Cluster 2                  %           from   %    ______________________________________     106! Translucent colony                   1                 99     107! Lipase   1                 99    ______________________________________                  Cluster 1          Cluster 4                  %                  %    ______________________________________    (none)    ______________________________________    C. Reference Number of unknown is ML201                       Percent in:                    Value in           Next    Character       unknown  Best Taxon                                       Best Taxon    ______________________________________     23! N-acetylglucosamine                        -        1       13     26! Saccharose     -        25      25     27! Maltose        -        50      25     32! Lactate        -        1       38     41! Propionate     -        1       1     43! Valerate       -        1       13     44! Citrate        -        50      13     45! Histidine      -        1       1     47! Glycogen       -        25      1     51! 3-Hydroxybutyrate                        -        1       13     52! 4-Hydroxybenzoate                        -        1       1     58! Leucine arylamidase                        +        50      88     59! Valine arylamidase                        +        25      88     64! Phosphohydrolase                        +        75      88     65! α-Galactosidase                        n.t.     75      1     85! Ampicillin     +        99      50     92! Fusidic Acid   +        99      25     93! Methicillin    +        99      50     99! Polymix        -        1       88     102!         Vancomycin     +        99      13     105!         Yellow colony  +        99      1     106!         Translucent colony                        n.t.     1       1     107!         Lipase         n.t.     1       1     108!         Oxidase (10 sec.)                        -        1       25    ______________________________________    n.t. = not tested    Isolate ML201 best identification is Cluster 5. Scores    for coefficients: 1 (Willcox probability), 2 (Taxonomic Dis-    tance), 3 (Standard Error of Taxonomic Distance).              1              2      3    ______________________________________    Cluster 5 0.9999         0.267  -0.176    Cluster 1 0.835 × 10.sup.-4                             0.437  2.435    Cluster 2 0.177 × 10.sup.-11                             0.641  7.593    ______________________________________                 Cluster 5    Characters against                   % in Taxon                             Value in unknown    ______________________________________    (none)    ______________________________________    Additional characters that assist in separating                  Cluster 5          Cluster 1                  %           from   %    ______________________________________    Galactosidase 75                  1    ______________________________________                  Cluster 5          Cluster 2                  %           from   %    ______________________________________    Galactosidase 75                  1     106! Translucent colony                   1                 99     107! Lipase   1                 99    ______________________________________    D. Reference Number of unknown is ML203                       Percent in:                    Value in           Next    Character       unknown  Best Taxon                                       Best Taxon    ______________________________________     23! N-acetylglucosamine                        +        26      20     26! Saccharose     +        74      20     27! Maltose        +        68      60     32! Lactate        +        99      99     41! Propionate     +        91      60     43! Valerate       +        97      80     44! Citrate        +        94      20     45! Histidine      +        71      1     47! Glycogen       +        26      20     51! 3-Hydroxybutyrate                        +        94      99     52! 4-Hydroxybenzoate                        +        71      80     58! Leucine arylamidase                        +        94      60     59! Valine arylamidase                        +        65      99     64! Phosphohydrolase                        -        3       20     65! α-Galactosidase                        n.t.     3       20     85! Ampicillin     -        56      1     92! Fusidic Acid   -        3       20     93! Methicillin    -        50      1     99! Polymix        +        81      99     102!         Vancomycin     +        3       20     105!         Yellow colony  -        1       1     106!         Translucent colony                        n.t.     3       1     107         Lipase         n.t.     21      1     108!         Oxidase (10 sec.)                        +        6       1    ______________________________________    n.t. = not tested    Isolate ML203 best identification is Cluster 3. Scores    for coefficients: 1 (Willcox probability), 2 (Taxonomic Dis-    tance), 3 (Stabdard Error of Taxonomic Distance).              1              2      3    ______________________________________    Cluster 3 0.9989         0.437  2.076    Cluster 4 0.1090 × 10.sup.-2                             0.526  4.590    Cluster 2 0.8137 × 10.sup.-9                             0.650  7.793    ______________________________________                 Cluster 3    Characters against                   % in Taxon                             Value in unknown    ______________________________________     102! Vancomycin                   3         +     108! Oxidase (10 sec.)                   6         +    ______________________________________    Additional characters that assist in separating                  Cluster 3          Cluster 4                  %           from   %    ______________________________________    (none)    ______________________________________    E. Reference Number of unknown is ML206                       Percent in:                    Value in           Next    Character       unknown  Best Taxon                                       Best Taxon    ______________________________________     23! N-acetylglucosamine                        -        1       13     26! Saccharose     +        25      25     27! Maltose        -        50      25     32! Lactate        -        1       38     41! Propionate     -        1       1     43! Valerate       -        1       13     44! Citrate        -        50      13     45! Histidine      -        1       1     47! Glycogen       +        25      1     51! 3-Hydroxybutyrate                        -        1       13     52! 4-Hydroxybenzoate                        -        1       1     58! Leucine arylamidase                        +        50      88     59! Valine arylamidase                        +        25      88     64! Phosphohydrolase                        +        75      88     65! α-Galactosidase                        n.t.     75      1     85! Ampicillin     +        99      50     92! Fusidic Acid   +        99      25     93! Methicillin    +        99      50     99! Polymix        -        1       88     102!         Vancomycin     +        99      13     105!         Yellow colony  +        99      1     106!         Translucent colony                        n.t.     1       1     107!         Lipase         n.t.     1       1     108!         Oxidase (10 sec.)                        -        1       25    ______________________________________    n.t. = not tested    Isolate ML206 best identification is Cluster 5. Scores    for coefficients: 1 (Willcox probability), 2 (Taxonomic Dis-    tance), 3 (Standard Error of Taxonomic Distance).              1              2      3    ______________________________________    Cluster 5 0.9999         0.345  1.766    Cluster 1 0.2530 × 10.sup.-5                             0.512  4.013    Cluster 6 0.2531 × 10.sup.-13                             0.652  10.883    ______________________________________                 Cluster 5    Characters against                   % in Taxon                             Value in unknown    ______________________________________    (none)    ______________________________________    Additional characters that assist in separating                  Cluster 5          Cluster 1                  %           from   %    ______________________________________    Galactosidase 75                  1    ______________________________________    F. Reference Number of unknown is ML301                       Percent in:                    Value in           Next    Character       unknown  Best Taxon                                       Best Taxon    ______________________________________     23! N-acetylglucosamine                        -        26      1     26! Saccharose     +        74      1     27! Maltose        +        68      1     32! Lactate        +        99      50     41! Propionate     +        91      99     43! Valerate       +        97      99     44! Citrate        +        94      50     45! Histidine      +        71      1     47! Glycogen       +        26      13     51! 3-Hydroxybutyrate                        +        94      25     52! 4-Hydroxybenzoate                        -        71      1     58! Leucine arylamidase                        +        94      63     59! Valine arylamidase                        +        65      25     64! Phosphohydrolase                        -        3       13     65! α-Galactosidase                        n.t.     3       1     85! Ampicillin     +        56      63     92! Fusidic Acid   -        3       1     93! Methicillin    +        50      13     99! Polymix        +        81      50     102!         Vancomycin     -        3       13     105!         Yellow colony  -        1       1     106!         Translucent colony                        n.t.     3       99     107!         Lipase         n.t.     21      99     108!         Oxidase (10 sec.)                        +        6       88    ______________________________________    n.t. = not tested    Isolate ML301 best identification is Cluster 3. Scores    for coefficients: 1 (Willcox probability), 2 (Taxonomic Dis-    tance), 3 (Standard Error of Taxonomic Distance).              1              2      3    ______________________________________    Cluster 3 1.000          0.429  1.918    Cluster 2 0.2841 × 10.sup.-6                             0.603  6.731    Cluster 4 0.9313 × 10.sup.-8                             0.623  6.704    ______________________________________                 Cluster 3    Characters against                   % in Taxon                             Value in unknown    ______________________________________     108! Oxidase (10 sec.)                   6         +    ______________________________________    Additional characters that assist in separating                  Cluster 3          Cluster 2                  %           from   %    ______________________________________     106! Translucent colony                   3                 99     107! Lipase  21                 99    ______________________________________

EXAMPLE 2 Production of Proteolytic Enzymes

Two alkaliphilic strains (1E.1 and 9B.1) were tested for the productionof proteolytic enzyme(s) in 7 different media poised at an alkaline pH.The experiments were carried out in 2 liter shake flasks with a baffle,each of the flasks contained 400 ml of the nutrient media R to X(Appendix A). The flasks were placed in an orbital incubator rotating atrevolutions per minute at a constant temperature of 37° C. Samples ofculture media were removed from the flasks at intervals of 1, 2, 3, 4,5, 6 and 8 days for the determination of enzyme content which isexpressed in Alkaline Delft Units (ADU--as described in British PatentSpecification 1,353,317).

Table 24 presents the maximum enzyme yields and the pH of thecultivation medium at the moment at which the measurement of enzymelevels were made.

                  TABLE 24    ______________________________________    Production of Proteolytic Enzymes           STRAIN 1E.1.sup.CT                         STRAIN 9B.1                       pH of             pH of    MEDIUM   ADU/ml    MEDIUM    ADU/ml  MEDIUM    ______________________________________    R        100       8.2       14      9.7    S        140       8.5       49      9.1    T        111       8.7       6       9.1    U         6        9.7       4       9.7    V         51       9.5       7       9.6    W         94       9.2       7       9.3    X        100       9.6       28      9.6    ______________________________________

The results of the test clearly indicate the presence of proteolyticenzymes, produced by the alkaliphilic bacteria of the present invention,in the culture broth.

EXAMPLE 3 Wash Performance Test Using Proteolytic Enzymes

Enzyme preparations from the alkaliphilic bacteria were tested in aspecially developed mini-wash test using cotton swatches (2.5×2.5 cm)soiled with milk, blood and ink (obtained from EMPA, St. Gallen,Switzerland, and designated EMPA 116). Prior to the wash test theswatches were pretreated with a solution containing an anionicsurfactant, sodium perborate and a bleach activator (TAED) at ambienttemperature for 15 minutes. After this treatment the test swatches wererinsed in running demineralized water for 10 minutes and air-dried. Thistreatment results in the fixation of the soil, making its removal moredifficult.

The washing tests were performed in 100 ml Erlenmeyer flasks providedwith a baffle and containing 30 ml of a defined detergent compositionplus 300 ADU protease to be tested. In each flask were placed twopre-treated EMPA 116 test swatches. The flasks were placed in areciprocal shaking water bath (2 cm stroke) and agitated at 320revolutions per minute. The tests were carried out at 40° C. for 30minutes. After washing, the swatches were rinsed in runningdemineralized water for 10 minutes and air-dried. The reflectance of thetest swatches was measured at 680 nm with a Photovolt photometer (Model577) equipped with a green filter.

The wash performance of the supernatant fraction of cultures of variousalkaliphilic bacteria in European powder detegents was determinedaccording to the method specified above. The supernatant fractions weresubjected to various treatments so as to produce enzyme-containingpreparations.

100 ml Erlenmeyer flasks were charged with powder detergent IECdissolved in standard tap water of 15° German Hardness so as to give afinal concentration of 4 g per liter.

The composition of the powder detergent IEC was as follows:

    ______________________________________    Component                wt %    ______________________________________    Linear sodium alkyl benzene sulphonate                             6.4    (mean chain length of alkane chain (C11.5))    Ethoxylated tallow alcohol (14EO)                             2.3    Sodium soap              2.8    Sodium tripolyphosphate (STPP)                             35.0    Sodium silicate          6.0    Magnesium silicate       1.5    Carboxy methyl cellulose 1.0    Sodium sulphate          16.8    Sodium perborate tetrahydrate                             18.5    TAED                     1.5    Miscellaneous + water    up to 100    ______________________________________     Standard tap water is composed of CaCl.sub.2.2H.sub.2 O, 0.291 g/l;     MgCl.6H.sub.2 O, 0.140 g/l and NaHCO.sub.3, 0.210 g/l dissolved in     demineralized water.

To each flask, two EMPA 116 swatches were added and sufficientenzyme-containing preparations to give a final activity of 300 ADU. Thefinal volume of the sud was 30 ml. By way of comparison, one flaskcontained no enzyme preparation, which was replaced with water. Thetrial was repeated either two or three times. The results are shown inTable 25.

                  TABLE 25    ______________________________________    Application Washing Trials    Performance of Proteolytic Enzyme-Containing Preparations    in a Washing Formulation.    Preparation             Average Remission of EMPA 116 Test Swatches    from Strain             Trial 1     Trial 2     Trial 3    ______________________________________    Untreated Culture Supernatant    None     11.4        11.8        13.0    (control)    1E.1.sup.CT             29.2        22.2        24.7    9B.1     23.7        23.9        24.2    17N.1.sup.CT         11.8        18.4    24B.1                17.3        16.3    Freeze Dried Supernatant Fraction    None     10.4        11.8        13.0    (control)    1E.1.sup.CT             15.1        28.9        30.0    9B.1     21.7        14.9        17.4    17N.1.sup.CT         13.7        17.9    24B.1                17.8        17.3    Dialyzed Supernatant Fractions    None     11.4        11.8        13.0    (control)    1E.1.sup.CT             26.4        22.7        26.3    9B.1     18.7        16.7        17.0    17N.1.sup.CT         12.0        12.6    24B.1                12.6        12.4    Ultrafiltration Concentrate of Supernatant Fractions    None     10.4        11.4    (control)    1E.1.sup.CT             14.6        26.0    9B.1     15.5        16.1    Acetone Precipitates of Supernatant Fractions    None     10.4        11.4    (control)    1E.1.sup.CT             13.4        23.4    9B.1     12.6        14.7    ______________________________________

The results of the trials demonstrate the efficacy of the proteolyticenzymes produced by the strains of the present invention, provided invarious forms, in detergent formulations and the improved washingperformance obtained.

EXAMPLE 4 Production of Starch Degrading Enzymes

Strain 1E.1^(CT) was tested for the production of starch degradingenzymes on a starch containing medium poised at an alkaline pH.

500 ml Erlenmeyer flasks were charged with 100 ml of alkaline medium(Medium Y, Appendix A) containing 2% soluble starch. The flasks wereinoculated (5%) with cells of strain 1E.1^(CT) grown for 24 hours onMedium A (37° C.). As controls, similar flasks of alkaline medium notcontaining starch were also inoculated.

The flasks were placed in an orbital shaking incubator rotating at 280revolutions per minute, at a constant temperature of 37° C. for 24hours. The fluid containing the enzyme activity was separated from thecells by centrifugation for 10 minutes at 4000 r.p.m.

The enzyme activity of the supernatant was determined using the reducingsugar assay of Nelson and Somogyi (Methods in Microbiology, volume 5B,pp. 300-301; (eds. J. R. Norris and D. W. Ribbons), Academic Press,London, 1971).

Determination of Starch Degrading Enzyme Activity by the Reducing SugarAssay Solutions

Reagent 1

144 g Na₂ SO₄ is dissolved by gentle warming in 500 ml demineralizedwater. 12 g potassium sodium tartrate tetrahydrate, 24 g Na₂ CO₃ and 16g NaHCO₃ are added to the solutions. The total volume of the solution isbrought to 800 ml by the addition of demineralized water.

Reagent 2

36 g Na₂ SO₄ is dissolved by gentle warming in 100 ml demineralizedwater and 4 g CuSO₄ ·5H₂ O is added to the warmed solution. The totalvolume of the solution is brought to 200 ml by the addition ofdemineralized water.

Directly before use, Reagents 1 and 2 are mixed in the ratio of 4:1(Reagent 1: Reagent 2).

Reagent 3

25 g ammonium molybdate tetrahydrate is dissolved in 450 mldemineralized water and 21 ml concentrated sulphuric acid is added withthorough mixing. 3 g Na₂ HAsO₄ ·7H₂ O are dissolved in 25 mldemineralized water and this solution is added to the molybdatesolution. The total solution is warmed for 48 hours at 37° C. and anyprecipitate is filtered off.

Standard

100 mg glucose is dissolved in demineralized water and the total volumeis brought to 100 ml. Before use, the solution is diluted 10 fold withdemineralized water.

Substrate

0.25% soluble starch (Merck, product number 1257) dissolved in 0.1M Na₂CO₃ -NaHCO₃ buffer, pH 10.1.

Assay

0.9 ml starch substrate solution, pH 10.1 is placed in a test-tube. Thetest-tube is placed in a water bath at 25° C. and allowed toequilibrate. The enzyme reaction is started by adding 0.1 ml of theenzyme-containing culture supernatant. The reaction is allowed toproceed for 30 minutes. The reaction is stopped by adding 1 ml ofReagent 1/2 and heating for 10 minutes at 100° C. The mixture is cooledon ice for 5 minutes and then 0.5 ml of Reagent 3 is added and the bluecolor is allowed to develop during 30 minutes at room temperature. Themixture is diluted by adding 1.0 ml demineralized water and theextinction is measured at 500 nm in a spectrophotometer. The reducingsugars are measured as glucose equivalents from a standard curve.

One unit of starch degrading enzyme activity is defined as 1 μg ofreducing sugars measured as glucose released per milliliter per minuteat pH 10.1 and 25° C.

The number of starch degrading enzyme units formed is shown in Table 26.

                  TABLE 26    ______________________________________    Production of Starch Degrading Enzymes by Strain 1E.1              OPTICAL              DENSITY      FINAL    ENZYME    MEDIUM    at 550 nm    pH       units per liter    ______________________________________    plus starch              2.25         9.4      1150    no starch 0.75         10.3      660    ______________________________________

The results of the test clearly indicate the presence of starchdegrading enzymes, produced by the alkaliphilic bacterial strain of thepresent invention, in the culture broth.

EXAMPLE 5 Stability of Starch Degrading Enzymes in Detergent

The ability of the starch degrading enzymes from strain 1E.1^(CT) towithstand detergents, which is essential for their application inlaundry detergents or textile desizing, is demonstrated.

100 ml Erlenmeyer flasks provided with a baffle were each charged with30 ml of 0.1M Na₂ CO₃ /NaHCO₃ buffer, pH 10.1 containing 0.12 g ofsodium dodecyl sulphate (equivalent to 4 g per liter). To one half ofthe flasks 0.3 g potato starch (equivalent to 1%) was added.

Each flask was dosed with enzyme-containing supernatant from strain1E.1^(CT) by adding 0.5, 1.0 or 2.0 ml (see Table 27). As a control, thesupernatant fluid was replaced with 1.0 ml water. Immediately afteradding the enzyme, a 0.1 ml sample was removed (time=zero hours) for themeasurement of enzyme activity.

The flasks were incubated with shaking at 25° C. for 2.5 hours at whichtime a second 0.1 ml sample was removed for the measurement of enzymeactivity.

As a comparison the experiment was repeated using a conventionalα-amylase derived from Bacillus subtilis.

Enzyme activity was determined using the reducing sugars methodpreviously described.

The results are recorded in Table 27.

                  TABLE 27    ______________________________________    Stability of Starch Degrading Enzymes from    Strain 1E.1.sup.CT in Detergents    ENZYME-    CONTAINING             ENZYME UNITS    SUPERNATENT            RECOVERED    ADDED (ml) CONDITIONS  pH      0 h.   2.5 h.    ______________________________________    0 *                    10.4     0      0    0.5        SDS         10.3    26     20    1.0                    10.3    44     48    2.0                    10.3    109    113    0 *                    10.3     0      0    0.5        SDS +       10.2    12     17    1.0        STARCH      10.1    36     48    2.0                    10.2    79     120    Standard § SDS                       10.4     0        0    Stabdard § SDS + STARCH                       10.2     0        0    ______________________________________     * replaced with 1 ml water     § 2.8 RAU Bacillus subtilis α-amylase  One RAU (reference     Amylase Unit) is defined as the quantity of enzyme that will convert 1 mg     of starch per minute at pH 6.6 and 30° C. into a product which upo     reaction with iodine has an equal absorbance at 620 nm. as a solution     containing 25 g CoCl.sub.2.6H.sub.2 O, 3.84 K.sub.2 Cr.sub.2 O.sub.7 and     ml 1 M HCl in 100 ml distilled water.

The results of this test clearly demonstrate the stability of the starchdegrading enzymes, produced by the alkaliphilic bacterial strain of thepresent invention, in the presence of detergent.

EXAMPLE 6 Production of Lipolytic Enzymes

Eleven of the new strains which clearly exhibited lipase activity(Appendix D) were tested further for the production of lipolyticenzymes. The eleven strains are examples from Cluster 2 and Cluster 3(FIG. 1).

The experiments were carried out in 100 ml conical flasks containing 30ml sterile alkaline nutrient medium, pH 9.6, inoculated with theappropriate bacterial strain. Three different media were used,designated medium Z to BB (Appendix A). The flasks were placed in anorbital shaking incubator (300 rpm) at 30° C. for 48 hours.

The cells were separated from the culture broth by centrifugation andthe supernatant dialyzed against 50 volumes 0.1 mM Tris-HCl buffer pH 9,with 3 changes of buffer over 24 hours. The dialysate was freeze driedto give a lipase preparation (Table 28).

The lipase preparations obtained according to this example were used forthe washing test described in Example 7, below.

                  TABLE 28    ______________________________________    Production of Lipase             PRODUCTION     LIPASE    LIPASE    STRAIN   MEDIUM         TLU/ml*   TLU/g    ______________________________________    39E.3    BB             1.3       134    40E.3    Z              1.2       118    41E.3    Z              1.1        82    42E.3    Z              1.2        76    44E.3    Z              1.2        99    45E.3.sup.CT             AA             1.4        98    48E.3    BB             2.0       152    49N.3    BB             1.5       123    50N.3    BB             2.0       100    51N.3    BB             1.0        98    52N.3    BB             1.2       128    ______________________________________     *TLU = True Lipase Unit as defined in U.S. Pat. No. 4,933,287.

The results of this test clearly demonstrate the presence of lipolyticenzymes, produced by alkaliphilic bacteria of the present invention, inthe culture broth and in a freeze-dried preparation of the dialyzedculture broth.

EXAMPLE 7 Lipase Washing Test

The lipase preparations from Example 6 were tested for performance underwashing conditions in TIDE^(R) powder (1.5 g/l), a detergent productfrom Procter & Gamble.

The washing test (SLM-test) was carried out as described in U.S. Pat.No. 4,933,287, which is hereby incorporated by reference. As control, alipase derived from Pseudomonas alcaligenes strain M1 (CB3 473·85) asdescribed in U.S. Pat. No. 4,933,287 was used. The results are shown inTable 29.

                  TABLE 29    ______________________________________    Lipase Washing Test    ______________________________________    Detergent: TIDE ® (powder), 1.5 g/l    Lipase:    2 TLU/ml    Ca.sup.2+ :               10.sup.-5 (sodium tripolyphosphate added)    pH:        9.5    ______________________________________             RECOVERY (%)               TYRIGLY-    STRAIN     CERIDES       TOTAL LIPID    ______________________________________    39E.3      55.3          73.4    40E.3      82.0          89.9    41E.3      44.7          78.7    42E.3      55.5          74.8    44E.3       6.6          76.9    45E.3.sup.CT               81.6          92.9    48E.3      76.5          82.9    49N.3      72.4          82.0    50N.3      50.5          76.6    51N.3      80.4          87.6    52N.2      77.0          84.7    M1         88.5          91.5    control*   98.7          98.7    ______________________________________     *Standard tap water as defined in U.S. Pat. No. 4,933,287

The decrease in the percent recovery of triglycerides and total lipids,as compared to the control, clearly indicate the ability of thelipolytic enzymes, produced by the alkaliphilic bacteria of the presentinvention, to break down and remove triglycerides and their degradationproducts embedded on a fabric sample, as well as their improvedperformance as compared to a known lipase.

                  APPENDIX A    ______________________________________    Media Used in the Present Invention    ______________________________________    MEDIUM A    Glucose                  10.0    gl.sup.-1    Peptone (Difco: Detroit, MI, USA)                             5.0     gl.sup.-1    Yeast Extract (Difco)    5.0     gl.sup.-1    K.sub.2 HPO.sub.4        1.0     gl.sup.-1    MgSO.sub.4.7H.sub.2 O    0.2     gl.sup.-1    NaCl                     40.0    gl.sup.-1    Na.sub.2 CO.sub.3        10.0    gl.sup.-1    *Agar                    20.0    gl.sup.-1    MEDIUM B    Glucose                  10.0    gl.sup.-1    Peptone (Difco)          5.0     gl.sup.-1    Yeast Extract (Difco)    5.0     gl.sup.-1    K.sub.2 HPO.sub.4        1.0     gl.sup.-1    MgSO.sub.4.7H.sub.2 O    0.2     gl.sup.-1    NaCl                     40.0    gl.sup.-1    Na.sub.2 CO.sub.3        10.0    gl.sup.-1    Novobiocin               50.0    mgl.sup.-1    Agar                     20.0    gl.sup.-1    MEDIUM C    Glucose                  10.0    gl.sup.-1    Peptone (Difco)          5.0     gl.sup.-1    Yeast Extract (Difco)    5.0     gl.sup.-1    K.sub.2 HPO.sub.4        1.0     gl.sup.-1    MgSO.sub.4.7H.sub.2 O    0.2     gl.sup.-1    NaCl                     40.0    gl.sup.-1    Na.sub.2 CO.sub.3        10.0    gl.sup.-1    Lactalbumin              10.0    gl.sup.-1    Agar                     20.0    gl.sup.-1    MEDIUM D    Glucose                  10.0    gl.sup.-1    Peptone (Difco)          5.0     gl.sup.-1    Yeast Extract (Difco)    5.0     gl.sup.-1    K.sub.2 HPO.sub.4        1.0     gl.sup.-1    MgSO.sub.4.7H.sub.2 O    0.2     gl.sup.-1    NaCl                     40.0    gl.sup.-1    Na.sub.2 CO.sub.3        10.0    gl.sup.-1    Casein                   20.0    gl.sup.-1    Agar                     20.0    gl.sup.-1    MEDIUM E    Soluble Starch           10.0    gl.sup.-1    Peptone (Difco)          5.0     gl.sup.-1    Yeast Extract (Difco)    5.0     gl.sup.-1    K.sub.2 HPO.sub.4        1.0     gl.sup.-1    MgSO.sub.4.7H.sub.2 O    0.2     gl.sup.-1    NaCl                     40.0    gl.sup.-1    Na.sub.2 CO.sub.3        10.0    gl.sup.-1    Lactalbumin              10.0    gl.sup.-1    Agar                     20.0    gl.sup.-1    MEDIUM F    Soluble Starch           10.0    gl.sup.-1    Peptone (Difco)          5.0     gl.sup.-1    Yeast Extract (Difco)    5.0     gl.sup.-1    K.sub.2 HPO.sub.4        1.0     gl.sup.-1    MgSO.sub.4.7H.sub.2 O    0.2     gl.sup.-1    NaCl                     40.0    gl.sup.-1    Na.sub.2 CO.sub.3        10.0    gl.sup.-1    Casein                   20.0    gl.sup.-1    Agar                     20.0    gl.sup.-1    MEDIUM G    Oxbile (Oxoid: Basingstoke, U.K.)                             10.0    gl.sup.-1    (NH.sub.4).sub.2 SO.sub.4                             5.0     gl.sup.-1    MgSO.sub.4.7H.sub.2 O    0.2     gl.sup.-1    Yeast Extract (Difco)    0.5     gl.sup.-1    Lactalbumin              10.0    gl.sup.-1    Agar                     20.0    gl.sup.-1    Adjusted to pH 8.5 with 50% Na.sub.2 CO.sub.3 solution    MEDIUM H    Oxbile (Oxoid)           10.0    gl.sup.-1    (NH.sub.4).sub.2 SO.sub.4                             5.0     gl.sup.-1    MgSO.sub.4.7H.sub.2 O    0.2     gl.sup.-1    Yeast Extract (Difco)    0.5     gl.sup.-1    Casein                   20.0    gl.sup.-1    Agar                     20.0    gl.sup.-1    Adjusted to pH 8.5 with 50% Na.sub.2 CO.sub.3 solution    MEDIUM I    Sabouroud Dextraose Agar (Oxoid)                             65.0    gl.sup.-1    NaCl                     40.0    gl.sup.-1    Na.sub.2 CO.sub.3        20.0    gl.sup.-1    Cycloheximide            20.0    gl.sup.-1    Penicillin G             25000   IUl.sup.-1    Streptomycin             25      mgl.sup.-1    MEDIUM J    Bacto Potato Dextrose Agar (Difco)                             39.0    gl.sup.-1    NaCl                     40.0    gl.sup.-1    Na.sub.2 CO.sub.3        20.0    gl.sup.-1    Novobiocin               50.0    mgl.sup.-1    MEDIUM K    Bacto Potato Dextrose Agar (Difco)                             39.0    gl.sup.-1    NaCl                     40.0    gl.sup.-1    Na.sub.2 CO.sub.3        20.0    gl.sup.-1    Cycloheximide            20.0    gl.sup.-1    Penicillin G             25000   IUl.sup.-1    Streptomycin             25.0    mgl.sup.-1    MEDIUM L    Glucose                  0.2     gl.sup.-1    Peptone (Difco)          0.1     gl.sup.-1    Yeast Extract (Difco)    0.1     gl.sup.-1    K.sub.2 HPO.sub.4        1.0     gl.sup.-1    MgSO.sub.4.7H.sub.2 O    0.2     gl.sup.-1    NaCl                     40.0    gl.sup.-1    Na.sub.2 CO.sub.3        10.0    gl.sup.-1    Casein                   20.0    gl.sup.-1    Agar                     20.0    gl.sup.-1    MEDIUM M (pH 9.6)    Oxbile (Oxoid)           2.0     gl.sup.-1    (NH.sub.4).sub.2 SO.sub.4                             1.0     gl.sup.-1    MgSO.sub.4.7H.sub.2 O    0.04    gl.sup.-1    Yeast Extract (Difco)    0.1     gl.sup.-1    Olive Oil                10.0    ml l.sup.-1    Na.sub.2 CO.sub.3        6.1     gl.sup.-1    Agar                     20.0    gl.sup.-1    MEDIUM N (pH 9.6)    Oxbile (Oxoid)           2.0     gl.sup.-1    (NH.sub.4).sub.2 SO.sub.4                             1.0     gl.sup.-1    MgSO.sub.4.7H.sub.2 O    0.04    gl.sup.-1    Yeast Extract (Difco)    0.1     gl.sup.-1    Olive Oil                10.0    ml l.sup.-1    Na.sub.2 CO.sub.3        6.1     gl.sup.-1    Tergitol 7 (Fluksa: Buchs, CH)                             500     ppm    Agar                     20.0    gl.sup.-1    MEDIUM O (pH 9.6)    Oxbile (Oxoid)           2.0     gl.sup.-1    (NH.sub.4).sub.2 SO.sub.4                             1.0     gl.sup.-1    MgSO.sub.4.7H.sub.2 O    0.04    gl.sup.-1    Yeast Extract (Difco)    0.1     gl.sup.-1    Olive Oil                10.0    ml l.sup.-1    Na.sub.2 CO.sub.3        6.1     gl.sup.-1    NaCl                     40.0    gl.sup.-1    Agar                     20.0    gl.sup.-1    MEDIUM P (pH 9.6)    Oxbile (Oxoid)           10.0    gl.sup.-1    (NH.sub.4).sub.2 SO.sub.4                             5.0     gl.sup.-1    MgSO.sub.4.7H.sub.2 O    0.2     gl.sup.-1    Yeast Extract (Difco)    0.5     gl.sup.-1    Olive Oil                10.0    ml l.sup.-1    NaCl                     40.0    gl.sup.-1    Agar                     20.0    gl.sup.-1    Adjusted to pH 9.6 with 50% Na.sub.2 CO.sub.3 solution    MEDIUM Q (pH 9.6a)    Oxbile (Oxoid)           10.0    gl.sup.-1    (NH.sub.4).sub.2 SO.sub.4                             5.0     gl.sup.-1    MgSO.sub.4.7H.sub.2 O    0.2     gl.sup.-1    Yeast Extract (Difco)    0.5     gl.sup.-1    Olive Oil                10.0    ml l.sup.-1    Agar                     20.0    gl.sup.-1    Adjusted to pH 9.6 with 50% Na.sub.2 CO.sub.3 solution    MEDIUM R (pH 9.5)    Fresh Yeast              82.5    gl.sup.-1    Glucose                  3.3     gl.sup.-1    K.sub.2 HPO.sub.4        1.6     gl.sup.-1    K.sub.2 CO.sub.3         0.6     gl.sup.-1    KHCO.sub.3               1.76    gl.sup.-1    CaCl.sub.2               0.05    gl.sup.-1    MgSO.sub.4.7H.sub.2 O    0.05    gl.sup.-1    FeSO.sub.4               0.005   gl.sup.-1    MnSO.sub.4               0.0066  gl.sup.-1    MEDIUM S    Fresh Yeast              8.25    gl.sup.-1    Glucose                  1.32    gl.sup.-1    K.sub.2 HPO.sub.4        1.6     gl.sup.-1    CaCl.sub.2               0.05    gl.sup.-1    MgSO.sub.4.7H.sub.2 O    0.05    gl.sup.-1    FeSO.sub.4               0.005   gl.sup.-1    MnSO.sub.4               0.0066  gl.sup.-1    NaCl                     40.0    gl.sup.-1    Adjusted to pH 10.5 with 40% Na.sub.2 CO.sub.3 solution    MEDIUM T (pH 10.1)    Glucose                  10.0    gl.sup.-1    Peptone (Difco)          5.0     gl.sup.-1    Yeast Extract (Difco)    5.0     gl.sup.-1    K.sub.2 HPO.sub.4        1.0     gl.sup.-1    MgSO.sub.4.7H.sub.2 O    0.2     gl.sup.-1    NaCl                     40.0    gl.sup.-1    Na.sub.2 CO.sub.3        10.0    gl.sup.-1    MEDIUM U    Oxbile                   10.0    gl.sup.-1    (NH.sub.4).sub.2 SO.sub.4                             5.0     gl.sup.-1    MgSO.sub.4.7H.sub.2 O    0.2     gl.sup.-1    Yeast extract (Difco)    0.5     gl.sup.-1    Casein                   10.0    gl.sup.-1    Adjusted to pH 9.8 with 40% Na.sub.2 CO.sub.3 solution    MEDIUM V    Tryptone Soya Broth (Oxoid)                             30.0    gl.sup.-1    Adjusted to pH 9.9 with 40% Na.sub.2 CO.sub.3 solution    MEDIUM W (pH 10.1)    Soluble starch           10.0    gl.sup.-1    Peptone (Difco)          5.0     gl.sup.-1    Yeast extract (Difco)    5.0     gl.sup.-1    KH.sub.2 PO.sub.4        1.0     gl.sup.-1    MgSO.sub.4.7H.sub.2 O    0.2     gl.sup.-1    NaCl                     40.0    gl.sup.-1    Na.sub.2 CO.sub.3        10.0    gl.sup.-1    MEDIUM X    Skim milk (Difco)        100.0   gl.sup.-1    Adjusted to pH 10.8 with 25% Na.sub.2 CO.sub.3 solution    MEDIUM Y    Yeast Extract (Difco)    1.0     g    KNO.sub.3                10.0    g    KH.sub.2 PO.sub.4        1.0     g    MgSO.sub.4.7H.sub.2 O    0.2     g    Na.sub.2 CO.sub.3        10.0    g    NaCl                     40.0    g    Soluble starch (Merck)   20.0    g    Demineralized water      1       liter    MEDIUM Z    Brain Heart Infusion (Difco)                             20.0    g    Na.sub.2 EDTA (Komplexion III, Siegfried AG,                             1.0     g    Switzerland)    FeSO.sub.4.7H.sub.2 O    0.006   g    MnSO.sub.4.H.sub.2 O     0.003   g    CaCl.sub.2.2H.sub.2 O    1.0     g    MgSO.sub.4.7H.sub.2 O    0.25    g    Tween 80                 5.0     g    Soya oil                 5.0     g    Distilled water to 1 liter, pH of medium    adjusted to pH 9.6 with 25% Na.sub.2 CO.sub.3 solution.    MEDIUM AA    Yeast Extract (Difco)    20.0    g    KH.sub.2 PO.sub.4        5.0     g    FeSO.sub.4.7H.sub.2 O    0.006   g    MnSO.sub.4.H.sub.2 O     0.003   g    CaCl.sub.2.2H.sub.2 O    1.0     g    MgSO.sub.4.7H.sub.2 O    0.25    g    Tween 80                 5.0     g    Soya oil                 5.0     g    Distilled water to 1 liter, pH of medium    adjusted to pH 9.6 with 25% Na.sub.2 CO.sub.3 solution.    MEDIUM BB    Brain Heart Infusion (Difco)                             20.0    g    FeSO.sub.4.7H.sub.2 O    0.006   g    MnSO.sub.4.H.sub.2 O     0.003   g    CaCl.sub.2.2H.sub.2 O    1.0     g    MgSO.sub.4.7H.sub.2 O    0.25    g    Tween 80                 5.0     g    Soya oil                 5.0     g    Distilled water to 1 liter, pH of medium    adjusted to pH 9.6 with 25% Na.sub.2 CO.sub.3 solution.    ______________________________________     *(when required for a solid medium)

APPENDIX B Methods for Unit Tests

1. Character numbers 1 to 5

Colony color, form, elevation, margin, size

A suspension of bacteria was spread over an alkaline nutrient agar(Medium A) and cultivated at 37° C. Colonies were examined after 48hours.

2. Character number 6 and 7

Cell morphology, Gram's strain reaction

Bacteria cells grown in alkaline nutrient broth (Medium A, without agar)for 24 hours were spun down in a centrifuge and resuspended in a smallamount of alkaline nutrient broth and allowed to air-dry on a microscopeslide. Or, bacteria were cultivated for 24-48 hours on an alkalinenutrient agar (Medium A) so as to form colonies. Colonies of bacteriawere suspended in physiological saline and a few drops allowed toair-dry on a microscope slide. The Gram's staining test was performedusing the Dussault modification (Journal of Bacteriology, 70, 484-485,1955) with safranin as counterstain.

3. Character number 8

Oxidase reaction

Filter paper moistened with a 1% aqueous solution ofN,N,N',N'-tetramethyl-p-phenylenediamine or oxidase identification discs(bioMerieux: Charbonieres-les-Bains, France) were smeared with a youngbacterial culture from alkaline nutrient agar. A purple color within 1minute was recorded as a positive reaction. E. coli, used as a control,did not give a positive reaction within one minute.

4. Character number 9

Skim milk test

A minimal medium composed (g/l distilled water) of yeast extract, 1.0;KNO₃, 10.0; K₂ HPO₄, 1.0; MgSO₄ ·7H₂ O, 0.2; NaCl 40.0; Na₂ CO₃, 10.0;agar, 20.0 was supplemented with 5.0 g/l skim milk powder, sterilised byautoclaving and poured into Petri dishes. Bacteria were inoculated andincubated at 37° C. Areas of clearing around bacterial colonies in anotherwise opaque agar were recorded as a positive reaction.Non-alkaliphilic reference strains were tested in an identical fashionusing media of the same composition but without Na₂ CO₃ so as to give apH of 6.8-7.0.

5. Character number 10

Gelatin hydrolysis

Charcoal-gelatin discs (bioMerieux) or "chargels" (Oxoid) were incubatedat 37° C. in an alkaline nutrient broth (Medium A) together withbacteria. A black sediment indicated a positive reaction.

6. Character number 11

NaCl tolerance

Two methods were applied.

(a) Bacterial strains were cultivated at 37° C. on an alkaline nutrientagar (Medium A) containing 0%, 4%, 8%, 12% or 15% (w/v) NaCl. The agarplates were examined for bacterial growth after 48 hours.

(b) Bacterial strains were cultivated at 37° C. in an alkaline nutrientbroth (Medium A) containing 0%, 4%, 8%, 12%, 15% or 25% NaCl. Bacterialgrowth was monitored by optical density measurements using a Klett meter(green filter) at 0, 12, 24, 48, 72 and 144 hours.

7. Character number 12

Minimum pH for growth

Nutrient agar, pH 6.8-7.0 (Medium A without sodium carbonate) was pouredinto square Petri dishes. A strip of solidified agar was removed fromone end and replaced with molten 4% (w/v) agar containing 3.6% (w/v) Na₂CO₃ and 0.8% (w/v) NaOH. A pH gradient from pH 10.5 to pH 7 across theplate was allowed to develop overnight. Bacteria were inoculated bystreaking along the pH gradient and cultivated at 37° C. for 48 hours.The pH at the point where bacterial growth ceased was measured with aflat head electrode and with "Alkalite" pH strips (Merck: Darmstadt, W.Germany).

8. Character numbers 13-21

Carbohydrate utilisation

A minimal medium composed (g/l distilled water) of yeast extract, 1.0;KNO₃, 10.0; K₂ HPO₄, 1.0; MgSO₄ ·7H₂ O, 0.2; NaCl, 40.0; Na₂ CO₃, 10.0;agar, 20.0 was supplemented with 2.0 g/l of the carbohydrate under testand poured into square Petri dishes. Bacteria were inoculated, using a25 point multipoint inoculator, from 1.0 ml of a bacterial suspensioncultivated for 48 hours in an alkaline nutrient broth (Medium A). Theagar-plates were incubated at 37° C. for 48 hours. The results wererecording by comparing bacterial growth on minimal nutrient mediumcontaining a carbohydrate supplement with growth on a minimal mediumwithout the carbohydrate under test. Non-alkaliphilic reference strainswere tested in an identical fashion using media of the same compositionbut without Na₂ CO₃ so as to give a pH of 6.8-7.0.

9. Character numbers 22-53

Growth on carbon substrates

Use was made of the commercially available test strip ATB 32 GN(API-bioMerieux: La Balme les Grottes, France). The strips were usedaccording to the manufacturer's instructions but with an addition of 1.0ml of a solution containing 4% NaCl and 1% Na₂ CO₃ to the vials of basalmedium provided. The strips were incubated at 37° C. for 48 hours.Non-alkaliphilic reference strains were incubated in the standard basalmedium.

10. Character numbers 54-72

Enzymatic activities

Use was made of the commercially available test strip APIZYM(API-bioMerieux) which was used according to the manufacturer'sinstructions, except that the alkaliphilic bacterial cells weresuspended in alkaline nutrient broth (Medium A). The strips wereincubated at 37° C. for 4 hours.

11. Character numbers 73-82

Amino acids as carbon and nitrogen source

The same technique was employed as for tests 14-21 except that KNO₃ wasomitted from the minimal nutrient medium.

12. Character numbers 83-104

Antibiotic sensitivity

A light suspension of bacteria in alkaline nutrient broth was spread onthe surface of alkaline nutrient agar (Medium A) and allowed to dry.Commercially available antibiotic susceptibility test discs (Oxoid orMast Laboratories: Merseyside, U.K.) were applied to the agar surface.The bacteria were cultivated at 37° C. for 48 hours. Clear zones aroundthe antibiotic disks indicated sensitivity.

                  APPENDIX C    ______________________________________    Unit Tests for Analysis by Numerical Taxonomy    CHAR-    ACTER   TEST               COMPUTER    NUMBER  DESCRIPTION        CODE    ______________________________________     1      Colony color       white = 1                               cream = 2                               beige = 3                               yellow = 4                               orange = 5                               pink = 6                               brown = 7                               red = 8     2      Colony form        circular = 1                               irregular = 2                               punctiform = 3                               filamentous = 4     3      Colony elevation   convex = 1                               raised = 2                               umbonate = 3                               flat = 4     4      Colony margin      entire = 1                               undulate = 2                               lobate = 3                               fimbriate = 4     5      Colony size        diameter in                               millimeters     6      Cell morphology    rod = 1                               coccus = 2     7      Gram's stain       negative = 1                               positive = 2     8      Oxidase test       negative = 1                               positive = 2     9      Skim milk test     negative = 1                               positive = 2    10      Gelatin hydrolysis negative = 1                               positive = 2    11      NaCl tolerance     growth at                               0%-4% = 1                               growth at                               0%-8% = 2                               growth at                               0%-12% = 3                               growth at                               0%-15% = 4                               growth only                               at 0% = 5                               growth only at                               4%-15% = 6    12      Minimum pH for     pH 7.5 = 7.5            growth on nutrient agar                               pH 8.0 = 8.0                               pH 8.5 = 8.5                               pH 9.0 = 9.0                               pH 9.5 = 9.5                               pH 10.0 = 10.0                               pH 10.5 =                               10.5    13-21   Carbohydrate utilisation    13      Formate    14      Fumarate    15      Succinate    16      Galactose          enhanced                               growth = 2    17      Pyruvate           equal growth = 1    18      Fructose           growth                               inhibitied = 0    19      Lactose    20      Xylose    21      Starch    22-53   Growth on carbon substrates    22      Rhamnose    23      N-acetylglucosamine    24      D-ribose    25      Inositol    26      D-saccharose    27      Maltose    28      Itaconate    29      Suberate    30      Malonate    31      Acetate    32      DL-lactate         positive = 2    33      L-alanine          negative = 1    34      Mannitol    35      D-glucose    36      Salicin    37      D-melibiose    38      L-fucose    39      D-sorbitol    40      L-arabinose    41      Propionate    42      Caprate    43      Valerate    44      Citrate    45      Histidine    46      5-ketogluconate    47      Glycogen    48      3-hydroxybenzoate    49      L-serine    50      2-ketogluconate    51      3-hydroxybutyrate    52      4-hydroxybenzoate    53      L-proline    54-72   Enzymatic activities    54      Alkaline phosphatase    55      Esterase (C4)    56      Esterase lipase (C8)    57      Lipase (C14)    58      Leucine arylamidase    59      Valine arylamidase    60      Cystine arylamidase                               positive = 2    61      Trypsin            negative = 1    62      Chymotrypsin    63      Acid phosphatase    64      Naphthol-AS-BI-phos-            phohydrolase    65      α-galactosidase    66      β-galactosidase    67      β-glucuronidase    68      α-glucosidase    69      β-glucosidase    70      N-acetyl-β-glycosaminidase    71      α-mannosidase    72      α-fucosidase    73-82   Amino acids as carbon and            nitrogen source    73      Serine    74      Proline    75      Asparagine    76      Arginine           enhanced                               growth = 2    77      Alanine            equal growth = 1    78      Lysine             no growth = 0    79      Methionine    80      Phenylalanine    81      Glycine    82      Valine     83-104 Antibiotic sensitivity    83      Gentamycin    10     μg    84      Nitrofurantoin                          50     μg    85      Ampicillin    25     μg    86      Nalidixic Acid                          30     μg    87      Sulphamethoxazole                          50     μg    88      Trimethoprim  2.5    μg    89      Penicillin G  1      IU  antibiotic sensitive    90      Chloramphenicol                          25     μg                                     inhibition of                                 growth =                                 2    91      Erythromycin  5      μg    92      Fusidic Acid  10     μg                                     antibiotic                                 insensitive,    93      Methicillin   10     μg                                     no growth                                 inhibition =                                 1    94      Novobiocin    5      μg    95      Streptomycin  10     μg    96      Tetracycline  25     μg    97      Sulphafurazole                          100    μg    98      Oleandomycin  5      μg    99      Polymixin     300    IU    100     Rifampicin    2      μg    101     Neomycin      30     μg    102     Vancomycin    30     μg    103     Kanamycin     30     μg    104     Bacitracin    10     IU    ______________________________________

    ______________________________________    Appendix D    Screening for Proteolytic, Amylolytic and Lipolytic Activity    ______________________________________    Proteolytic Activity    STRAIN LACTALBUMIN   CASEIN   BLOOD  GELATIN    ______________________________________    Cluster 1    1E.1.sup.CT           +             +        +      +    2E.1   -             -        -      -    wB2    -             -        -      +    wB5    -             -        -      -    wBs4   -             +        +      +    10B.1  +             +        +      +    20N.1  +             +        -      +    27M.1  -             +        -      -    wNk2   -             +        -      +    Cluster 2    39E.3  n.t.          n.t.     n.t.   +    41E.3  n.t.          n.t.     n.t.   +    45E.3.sup.CT           n.t.          n.t.     n.t.   +    47E.3  n.t.          n.t.     n.t.   +    51N.3  n.t.          n.t.     n.t.   +    52N.3  n.t.          n.t.     n.t.   +    42E.3  n.t.          n.t.     n.t.   +    50N.3  n.t.          n.t.     n.t.   +    Cluster 3    6B.1   +             +        -      +    7B.1   +             +        -      -    8B.1   -             +        -      +    38E.2  n.t.          n.t.     n.t.   -    56E.4  +             n.t.     n.t.   -    25B.1  n.t.          +        n.t.   +    26N.1  -             +        -      +    11C.1  +             +        -      +    wB.1   -             -        -      +    12C.1  -             +        -      -    28N.1.sup.CT           -             -        -      -    61N.4  +             n.t.     n.t.   -    36E.2  n.t.          n.t.     n.t.   -    40E.3  n.t.          n.t.     n.t.   +    65B.4  +             n.t.     n.t.   -    94LM.4 n.t.          n.t.     n.t.   +    19N.1  -             +        -      +    24B.1  +             +        -      +    21M.1  +             +        +      +    29C.1  -             -        -      -    35E.2  n.t.          n.t.     n.t.   -    37E.2  n.t.          n.t.     n.t.   -    48E.3  n.t.          n.t.     n.t.   +    78LN.4 n.t.          +        n.t.   +    73aC.4 n.t.          +        n.t.   +    75C.4  n.t.          +        n.t.   +    73bC.4 n.t.          +        n.t.   +    74C.4  n.t.          +        n.t.   -    77LN.4 n.t.          +        n.t.   -    wN1    -             -        -      -    49N.3  n.t.          n.t.     n.t.   +    44E.3  n.t.          n.t.     n.t.   +    58E.4  n.t.          n.t.     n.t.    57E.4  +             n.t.     n.t.   +    Cluster 4    wE5    -             -        -      +    wB4.sup.CT           -             -        -      -    wNk1   -             -        -      +    wE11   -             -        -      -    wE12   -             -        -      +    Cluster 5    9B.1   -             +        -      +    16N.1  +             n.t.     n.t.   +    17N.1.sup.CT           +             +        -      +    22M.1  +             +        -      +    Cluster 6    18N.1  +             +        -      +    59E.4  +             n.t.     n.t.   +    64B.4.sup.CT           +             n.t.     n.t.   +    63N.4  +             n.t.     n.t.   +    53E.4  +             n.t.     n.t.   +    Non-Clustering Strains    wN.2   -             -        -      +    4E.1   -             -        -      -    5E.1   -             -        -      +    92LM.4 n.t.          n.t.     n.t.   +    wBn5   -             -        -      +    ______________________________________    Amylolytic and Lipolytic Activity*           STARCH     LIPASE AC- ESTERASE LIPASE           HYDROL-    TIVITY ON  LIPASE   ACTIV-    STRAIN YSIS       OLIVE OIL  ACTIVITY ITY    ______________________________________    Cluster 1    1E.1.sup.CT           +          -          +        +    2E.1   +          -          +        -    wB2    -          -          +        -    wB5    -          -          +        -    wBs4   -          -          +        -    10B.1  +          -          +        -    20N.1  -          -          +        -    27M.1  -          -          +        -    wNk2   +          -          +        -    Cluster 2    39E.3  +          +          +        -    41E.3  +          +          +        -    45E.3.sup.CT           +          +          +        +    47E.3  +          +          -        -    51E.3  +          +          +        +    52E.3  +          +          +        +    42E.3  +          +          +        +    50N.3  +          +          +        +    Cluster 3    6B.1   -          -          +        -    7B.1   +          -          +        +    8B.1   +          +          +        +    38E.2  -          -          +        -    56E.4  -          +          +        +    25B.1  -          -          +        +    26N.1  -          -          +        -    11C.1  -          -          +        -    wB1    -          -          +        -    12C.1  +          -          -        -    28N.1.sup.CT           +          -          +        -    61N.4  +          -          -        -    36E.2  -          -          -        -    40E.3  +          +          +        +    65B.4  +          -          +        -    94LM.4 +          -          +        -    19N.1  +          -          +        +    24B.1  -          -          +        -    21M.1  +          -          -        -    29C.1  +          -          +        +    35E.2  -          -          +        -    37E.2  -          -          +        -    48E.3  -          +          +        -    78LN.4 -          -          +        -    73aC.4 -          -          +        -    75C.4  +          -          -        -    73bC.4 +          -          +        -    74C.4  +          -          +        -    77LN.4 -          -          +        +    wN1    +          -          +        -    49N.3  +          +          +        -    44E.3  +          +          +        +    58E.4  +          +          +        -    57E.4  +          -          +        -    Cluster 4    wE5    +          -          -        -    wB4.sup.CT           -          -          +        -    wNk1   +          -          +        -    wE11   -          -          +        -    wE12   -          -          +        -    Cluster 5    9B.1   +          -          +        -    16N.1  +          -          +        -    17N.1.sup.CT           +          -          +        -    22M.1  +          -          +        -    Cluster 6    18N.1  +          -          +        -    59E.4  +          -          +        -    64B.4.sup.CT           +          -          +        -    63N.4  +          -          -        -    53R.4  +          -          +        -    Non-Clustering Strains    wN2    +          -          +        +    4E.1   -          -          +        -    5E.1   +          -          +        -    92LM.4 +          +          +        -    wBn5   -          -          +        -    ______________________________________     n.t. = not tested     *Starch Hydrolysis determined according to Character 21 (Appendix B)     Lipase Activity (Olive Oil) determined on media M-P (Appendix A)     Esterase Lipase Activity determined according to Character 56 (Appendix B     Lipase Activity determined according to Character 57 (Appendix B)

    ______________________________________    Appendix E    Percentage Positive States for Characters in Clusters                 CLUSTER    CHARACTER      1      2      3    4    5    6    ______________________________________     6!  Cell morphology                       9      0    12   0    0    0     7!  Gram's stain  0      0    0    0    0    0     8!  Oxidase test  55     100  35   80   0    20     9!  Skim milk test                       36     11   6    20   0    20     10! Gelatin       67     100  56   60   100  100     13! Formate       0      0    15   40   0    0     16! Galactose     45     11   12   40   25   0     17! Pyruvate      64     89   88   40   75   0     18! Fructose      45     11   68   60   50   100     19! Lactose       9      0    0    0    0    0     20! Xylose        18     11   15   20   0    0     21! Starch        73     100  91   20   100  100     22! Phamnose      9      0    15   20   0    0     23! N-actylglu-   9      0    26   20   0    100         cosamine     24! D-ribose      27     0    50   0    0    20     25! Inositol      9      0    9    0    50   0     26! D-saccharose  27     0    74   20   25   100     27! Maltose       9      11   74   40   50   100     28! Itaconate     0      11   6    0    0    0     29! Suberate      9      67   53   40   0    0     30! 0             11     65   0    25   40     31! Acetate       18     100  100  80   25   100     32! DL-lactate    27     56   100  100  0    40     33! L-alanine     18     89   82   100  0    100     34! Mannitol      0      0    41   20   0    80     35! D-glucose     9      11   71   60   0    60     36! Salicin       0      0    3    20   0    60     37! D-melibiose   0      0    3    0    0    0     38! L-fucose      0      0    0    20   0    20     39! D-sorbitol    18     0    41   0    0    40     40! L-arabinose   9      0    3    40   0    0     41! Propionate    9      100  94   80   0    80     42! Caprate       0      78   32   40   0    80     43! Valerate      9      100  97   80   0    40     44! Citrate       9      56   94   20   50   100     45! Histidine     0      0    71   0    0    80     46! 5-ketogluconate                       0      0    21   0    0    0     47! Glycogen      9      22   26   20   25   100     48! 3-hydroxybenzoate                       9      0    38   0    0    0     49! L-serine      0      0    68   60   0    0     50! 2-ketogluconate                       0      0    56   80   25   20     51! 3-hydroxybutyrate                       18     33   94   100  0    100     52! 4-hydroxybenzoate                       0      0    71   80   0    0     53! L-proline     45     100  100  80   25   80     54! Alkaline      100    44   94   40   100  60         phosphatase     55! Esterase (C4) 100    100  100  100  100  100     56! Esterase lipase (C8)                       100    89   85   100  100  100     57! Lipase (C14)  9      67   26   0    0    0     58! Leucine arylamidase                       91     67   94   60   50   0     59! Valine arylamidase                       91     33   65   100  25   0     60! Cystine arylamidase                       73     0    15   0    0    0     61! Trypsin       64     11   9    60   0    0     62! Chymotrpsin   73     0    3    20   75   0     63! Acid phosphatase                       91     11   94   100  100  60     64! Naphthol, phospho-                       91     22   3    20   100  40         hydrolase     65! α-galactosidase                       0      11   3    20   100  0     66! β-galactosidase                       9      0    0    20   100  0     67! β-glucuronidase                       9      0    3    20   25   0     68! α-glucosidase                       27     0    79   60   100  40     69! β-glucosidase                       9      0    9    80   75   0     70! N-acetyl-β-                       27     0    0    0    0    0         glucosaminidase     71! α-mannosidase                       0      0    0    0    0    0     72! α-fucosidase                       9      0    0    20   0    0     73! Serine        22     13   29   60   100  50     74! Proline       56     63   65   80   100  100     75! Asparagine    67     38   61   60   75   100     76! Arginine      56     25   53   80   50   50     77! Alanine       67     100  62   40   100  50     78! Lysine        44     75   71   80   75   100     79! Methionine    60     50   53   nc   50   50     80! Phenylalanine 89     100  76   100  100  100     81! Glycine       44     13   29   80   50   50     82! Valine        44     50   41   0    50   25     83! Gentamycin    36     67   3    20   0    0     84! Nitrofurantoin                       18     33   21   0    0    0     85! Ampicillin    45     67   56   0    100  80     86! Nalidixic Acid                       36     78   76   0    0    20     87! Sulphamethoxazole                       18     33   38   0    0    0     88! Trimethoprim  45     22   35   0    75   60     89! Penicillin G  27     11   29   0    75   100     90! Chloramphenicol                       100    100  100  100  100  100     91! Erythromycin  91     100  100  100  100  100     92! Fusidic Acid  18     0    3    20   100  100     93! Methicillin   45     11   50   0    100  100     94! Novobiocin    18     0    3    0    25   0     95! Streptomycin  100    78   97   100  75   100     96! Tetracycline  45     22   3    20   100  100     97! Sulphafurazole                       40     13   20   nc   0    0     98! Oleandomycin  100    38   94   100  100  100     99! Polymixin     89     63   78   100  0    0     100!         Rifampicin    100    38   78   100  100  100     101!         Neomycin      0      13   0    0    0    0     102!         Vancomycin    22     13   0    20   100  75     103!         Kanamycin     11     13   0    0    0    0     104!         Bacitracin    33     13   0    20   100  100    ______________________________________

We claim:
 1. A substantially pure preparation of alkali-tolerantenzymes, prepared by culturing bacteria in a culture medium, separatingthe bacteria from the culture medium, and recovering enzyme activityfrom the culture medium, wherein said bacteria are a pure bacterialculture consisting of aerobic, Gram-negative, rod-shaped, obligatealkaliphilic bacteria having the following characteristics:a) formcream-colored, circular colonies; b) grow optimally between pH 9 and pH10; c) give a positive response to the following tests:1) Leucinearylamidase 2) Valine arylamidase 3) Phosphohydrolase 4) Polymixin; d)give a negative response to the following tests:1) N-acetylglucosamine2) Maltose 3) Propionate 4) Caprate 5) Valerate 6) Citrate 7) Histidine8) Glycogen 9) 4-hydroxybenzoate 10) α-galactosidase,wherein the enzymeshave an activity selected from the group consisting of proteolytic,lipolytic and starch degrading activities.
 2. A substantially purepreparation of alkali-tolerant enzymes, prepared by culturing bacteriain a culture medium, separating the bacteria from the culture medium,and recovering enzyme activity from the culture medium, wherein saidbacteria are a pure bacterial culture consisting of aerobic,Gram-negative, rod-shaped, obligate alkaliphilic bacteria having thefollowing characteristics:a) form small, cream-colored colonies; b) growoptimally between pH 7.8 and pH 11.2; c) give a positive response to thefollowing tests:1) Starch 2) Acetate 3) Propionate 4) Valerate 5)Proline 6) Lipase 7) Oxidase (response within 10 seconds); d) give anegative response to the following tests:1) N-acetylglucosamine 2)Saccharose 3) Histidine 4) 2-ketogluconate 5) 4-hydroxybenzoate 6)α-glucosidase 7) β-glucosidase 8) Fusidic Acid,wherein the enzymes havean activity selected from the group consisting of proteolytic, lipolyticand starch degrading activities.
 3. A substantially pure preparation ofalkali-tolerant enzymes, prepared by culturing bacteria in a culturemedium, separating the bacteria from the culture medium, and recoveringenzyme activity from the culture medium, wherein said bacteria are apure bacterial culture consisting of aerobic, Gram-negative, rod-shaped,obligate alkaliphilic bacteria having the following characteristics:a)form cream-colored, opaque colonies; b) grow optimally between pH 8.5and pH 10.7; c) contains ubiquinone 6 as a major respiratory quinone; d)give a positive response to the following tests:1) Acetate 2) Lactate 3)Propionate 4) Valerate 5) Citrate 6) 3-hydroxybenzoate 7) Proline 8)Leucine arylamidase; e) give a negative response to the followingtests:1) Phosphohydrolase 2) α-galactosidase 3) Fusidic Acid 4)Tetracycline 5) Vancomycin 6) Bacitracin,wherein the enzymes have anactivity selected from the group consisting of proteolytic, lipolyticand starch degrading activities.
 4. A substantially pure preparation ofalkali-tolerant enzymes, prepared by culturing bacteria in a culturemedium, separating the bacteria from the culture medium, and recoveringenzyme activity from the culture medium, wherein said bacteria are purebacterial culture consisting of aerobic, Gram-negative, rod-shaped,obligate alkaliphilic bacteria having the following characteristics:a)form beige to brown-colored, opaque colonies; b) grow optimally betweenpH 7.5 and pH 10.9; c) contains ubiquinone 9 as a major respiratoryquinone; d) give a positive response to the following tests:1) Lactate2) Alanine 3) 3-hydroxybutyrate 4) Valine arylamidase 5) Polymixin; e)give a negative response to the following tests:1) Histidine 2)Ampicillin 3) Naladixic acid 4) Trimethoprim 5) Penicillin G 6)Methicillinwherein the enzymes have an activity selected from the groupconsisting of proteolytic, lipolytic and starch degrading activities. 5.A substantially pure preparation of alkali-tolerant enzymes, prepared byculturing bacteria in a culture medium, separating the bacteria from theculture medium, and recovering enzyme activity from the culture medium,wherein said bacteria are a pure bacterial culture consisting ofaerobic, Gram-negative, rod-shaped, obligate alkaliphilic bacteriahaving the following characteristics:a) form bright yellow-coloredcolonies; b) grow-optimally between pH 8 and pH 10.5; c) give a positiveresponse to the following tests:1) Phosphohydrolase 2) α-galactosidase3) β-galactosidase 4) Ampicillin 5) Fusidic Acid 6) Methicillin 7)Tetracycline 8) Vancomycin 9) Bacitracin; d) give a negative response tothe following tests:1) N-acetylglucosamine 2) Lactate 3) L-alanine 4)Mannitol 5) Propionate 6) Caprate 7) Valerate 8) Histidine 9)3-hydroxybenzoate 10) 3-hydroxybutyrate 11) 4-hydroxybenzoate 12)Polymixinwherein the enzymes have an activity selected from the groupconsisting of proteolytic, lipolytic and starch degrading activities. 6.A substantially pure preparation of alkali-tolerant enzymes, prepared byculturing bacteria in a culture medium, separating the bacteria from theculture medium, and recovering enzyme activity from the culture medium,wherein said bacteria are a pure bacterial culture consisting ofaerobic, Gram-negative, rod-shaved, obligate alkaliphilic bacteriahaving the following characteristics:a) form cream to beige-colored,irregular, flat colonies; b) grow optimally between pH 8.2 and pH 10.9;c) give a positive response to the following tests:1) Starch 2)N-acetylglucosamine 3) Saccharose 4) Maltose 5) Acetate 6) Alanine 7)Citrate 8) Glycogen 9) 3-hydroxybutyrate 10) Penicillin G 11) FusidicAcid 12) Methicillin 13) Tetracycline 14) Bacitracin; d) give a negativeresponse to the following tests:1) Pyruvate 2) 4-hydroxybenzoate 3)Leucine arylamidase 4) Valine arylamidase 5) α-galactosidase 6)Polymixinwherein the enzymes have an activity selected from the groupconsisting of proteolytic, lipolytic and starch degrading activities. 7.A substantially pure preparation of alkali-tolerant enzymes, prepared byculturing bacteria in a culture medium, separating the bacteria from theculture medium, and recovering enzyme activity from the culture medium,wherein said bacteria are a pure bacterial culture consisting ofaerobic, Gram-negative, rod-shaped, obligate alkaliphilic bacteriahaving the following characteristics:a) cells frequently in pairs; b)grow optimally between pH 9 and pH 10; c) on alkaline agar, form smooth,translucent, beige colored colonies, 1-2 mm in diameter which arecircular, convex with an entire margin; d) in alkaline broth growth at37° C. is flocculent with a ring or surface pellicle and formation of asediment; e) grow optimally at 20° C. to 30° C.; f) do not grow at 15°C. or 40° C.; g) KOH test is positive; h) aminopeptidase test is weakpositive; i) oxidase test is weak positive; j) catalase test ispositive; k) obligate halophile; l) grow optimally at 4% NaCl; m) do notgrow at 0% or 8% NaCl; n) hydrolysis of gelatin test is slow positive;o) hydrolysis of starch is positive; p) does not grow on simple sugars;q) does not grow on organic acids; r) grow on yeast extract andpeptoneswherein the enzymes have an activity selected from the groupconsisting of proteolytic, lipolytic and starch degrading activities. 8.A substantially pure preparation of alkali-tolerant enzymes, prepared byculturing bacteria in a culture medium, separating the bacteria from theculture medium, and recovering enzyme activity from the culture medium,wherein said bacteria are a pure bacterial culture consisting ofaerobic, Gram-negative, rod-shaped, obligate alkaliphilic bacteriahaving the following characteristics:a) grow optimally between pH 8.2and pH 10.9; b) on alkaline agar, form smooth, opaque, beige or browncolored colonies, 2-4 mm in diameter which are circular in form, convexin elevation, with an entire margin; c) in alkaline broth, growth at 37°C. is heavy and flocculent with a sediment and surface pellicle; d) growoptimally between 20° C. and 37° C.; e) do not grow at 8° C. or at 40°C. or above; f) KOH test is positive; g) aminopeptidase test ispositive; h) oxidase test is very weakly positive; i) catalase test ispositive; j) grow at a NaCl concentration of between 0% and 15%; k) donot grow at 20% NaCl; l) hydrolysis of gelatin test is negative; m)hydrolysis of starch is negative; n) grow on yeast extract; o) grow onorganic acids selected from the group consisting of succinate, pyruvate,citrate, malonate, acetate and lactate; p) grow on fatty acids selectedfrom the group consisting of propionate, valerate and suberate; q) growon amino acids selected from the group consisting of proline, serine,histidine and lysinewherein the enzymes have lipolytic activity.
 9. Asubstantially pure preparation of alkali-tolerant enzymes, prepared byculturing bacteria in a culture medium, separating the bacteria from theculture medium, and recovering enzyme activity from the culture medium,wherein said bacteria are a pure bacterial culture consisting ofaerobic, Gram-negative, rod-shaped, obligate alkaliphilic bacteriahaving the following characteristics:a) grow optimally between pH 9 andpH 10.5; b) on alkaline agar, form-smooth, opaque, brown coloredcolonies, 3-4 mm in diameter which are fairly irregular in form,generally flat to slightly umbonate in elevation with a lobate margin;c) in alkaline broth, growth at 37° C. is moderate to heavy, becomingflocculent with a sediment and surface pellicle; d) grow optimallybetween 20° C. and 40° C.; e) do not grow at 45° C.; f) KOH test ispositive; g) aminopeptidase test is positive; h) oxidase test isnegative; i) catalase test is positive; j) grow at a NaCl concentration0% to 12%; k) do not grow at 20% NaCl; l) hydrolysis of gelatin test ispositive; m) hydrolysis of starch is weakly positive; n) does not growon simple sugars; o) grow on yeast extract; p) grow on organic acidsselected from the group consisting of pyruvate, citrate, acetate andlactate; q) grow on fatty acids selected from the group consisting ofpropionate, caprate and valerate; r) grow on amino acids selected fromthe group consisting of proline, alanine and lysinewherein the enzymeshave an activity selected from the group consisting of proteolytic,lipolytic and starch degrading activities.
 10. A substantially purepreparation of alkali-tolerant enzymes, prepared by culturing bacteriain a culture medium, separating the bacteria from the culture medium,and recovering enzyme activity from the culture medium, wherein saidbacteria are a pure bacterial culture consisting of aerobic,Gram-negative, rod-shaped, obligate alkaliphilic bacteria having thefollowing characteristics;a) does not grow below pH 7.5; b) on alkalineagar, form smooth, cream colored colonies, initially translucent butbecoming opaque; c) on alkaline agar, the colonies develop fromcircular, entire and become irregular, lobate in form, with a convexelevation; d) in alkaline broth, growth at 37° C. is slow, slight,flocculent with a sediment but no surface pellicle; e) grow optimallybetween 10° C. and 40° C.; f) do not grow at 8° C. or 45° C.; g) KOHtest is positive; h) aminopeptidase test is negative; i) oxidase test ispositive; j) catalase test is positive; k) grow at an NaCl concentrationof between 0% to 15%; l) do not grow at 20% NaCl; m) hydrolysis ofgelatin test is positive; n) hydrolysis of starch is weakly positive; o)grow on yeast extract and peptones; p) grow on sugars; q) grow onorganic acids; r) grow on amino acidswherein the enzymes have anactivity selected from the group consisting of proteolytic, lipolyticand starch degrading activities.
 11. A substantially pure preparation ofalkali-tolerant enzymes, prepared by culturing bacteria in a culturemedium, separating the bacteria from the culture medium, and recoveringenzyme activity from the culture medium, wherein said bacteria are apure bacterial culture consisting of aerobic, Gram-negative, rod-shaped,obligate alkaliphilic bacteria having the following characteristics:a)cells frequently form short chains; b) does not grow below pH 8; c) onalkaline agar, form smooth, circular, convex colonies with an entiremargin, about 1 mm in diameter which are initially transparent, creambeige in color, the colonies become opaque and brown in color with age;d) in alkaline broth, growth at 37° C. is initially evenly turbid with asediment but no surface pellicle becoming flocculent with formation of apellicle; e) grow optimally between 30° C. and 37° C.; f) do not grow at40° C.; g) KOH test is positive; h) aminopeptidase test is positive; i)oxidase test is positive; j) catalase test is positive; k) obligatehalophile; l) grow at 4% NaCl; m) do not grow at 0% or 8% NaCl; n)hydrolysis of gelatin test is slow positive; o) hydrolysis of starch isnegative; p) grow on yeast extract and peptones; q) grow on sugars; r)grow on organic acids; s) grow on fatty acids; t) grow on aminoacidswherein the enzymes have an activity selected from the groupconsisting of proteolytic and starch degrading activities.